Heterocyclic protein kinase inhibitors

ABSTRACT

The present invention provides protein kinase having one of the following structures (I), (II) or (III): or a stereoisomer, prodrug, tautomer or pharmaceutically acceptable salt thereof, wherein R, R 1 , R 2  and X are as defined herein. Compositions and methods for using the same in the treatment of cancer, autoimmune, inflammatory and other Pim kinase-associated conditions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application No. 61/510,207, filed on Jul. 21,2011; U.S. Provisional Patent Application No. 61/632,834, filed on Jul.21, 2011; U.S. Provisional Patent Application No. 61/632,826, filed onMar. 1, 2012; and U.S. Provisional Patent Application No. 61/608,028,filed on Mar. 7, 2012; which applications are each incorporated hereinby reference in their entireties.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates, in general, to compounds that inhibitprotein kinase activity, and to compositions and methods relatedthereto.

2. Description of the Related Art

Cancer (and other hyperproliferative diseases) is characterized byuncontrolled cell proliferation. This loss of the normal control of cellproliferation often appears to occur as the result of genetic damage tocell pathways that control progress through the cell cycle. The cellcycle consists of DNA synthesis (S phase), cell division or mitosis (Mphase), and non-synthetic periods referred to as gap 1 (G1) and gap 2(G2). The M-phase is composed of mitosis and cytokinesis (separationinto two cells). All steps in the cell cycle are controlled by anorderly cascade of protein phosphorylation and several families ofprotein kinases are involved in carrying out these phosphorylationsteps. In addition, the activity of many protein kinases increases inhuman tumors compared to normal tissue and this increased activity canbe due to many factors, including increased levels of a kinase orchanges in expression of co-activators or inhibitory proteins.

Cells have proteins that govern the transition from one phase of thecell cycle to another. For example, the cyclins are a family of proteinswhose concentrations increase and decrease throughout the cell cycle.The cyclins turn on, at the appropriate time, different cyclin-dependentprotein kinases (CDKs) that phosphorylate substrates essential forprogression through the cell cycle. Activity of specific CDKs atspecific times is essential for both initiation and coordinated progressthrough the cell cycle. For example, CDK1 is the most prominent cellcycle regulator that orchestrates M-phase activities. However, a numberof other mitotic protein kinases that participate in M-phase have beenidentified, which include members of the polo, aurora, and NIMA(Never-In-Mitosis-A) families and kinases implicated in mitoticcheckpoints, mitotic exit, and cytokinesis.

Pim kinases (e.g., Pim-1 kinase, Pim-2 kinase, Pim-3 kinase) are afamily of oncogenic serine/threonine kinases. Pim-1 kinase is known tobe involved in a number of cytokine signaling pathways as a downstreameffector. Once activated, Pim-1 kinase causes progression of the cellcycle, inhibition of apoptosis and modulation of other signaltransduction pathways, including its own. Pim-1 kinase is also known toeffect activation of transcription factors such as NFAT, p100, c-Myb andPap-1, and inhibition of others such as HP1. Normal expression of Pim-1kinase is seen in cells of hematopoietic origin, such as fetal liver,thymus, spleen and bone marrow. Additionally, expression is seen inprostate and oral epithelial cells Pim-1 kinase is believed to beinvolved in the initiation or progression of malignant transformationleading to malignancies including Burkitt's lymphoma, prostate cancer,oral cancer and diffuse large cell lymphomas, among others.

Pim kinases also play a role in immune regulation. For example, enhancedPim expression has been observed in a variety of inflammatory states.Pim-2 is also implicated in cytokine induced T-cell growth and survival.A recent publication (Jackson et al., Cell Immunology, 2012, 272,200-213) demonstrated in vivo efficacy for a dual PIM-1 and PIM-3inhibitor in a mouse inflammatory bowel disease model. Therefore, PIMkinases are attractive targets for various autoimmune and/orinflammatory diseases.

Based on their involvement in a number of human malignancies, there is aneed for the rational design of specific and selective inhibitors forthe treatment of cancer and other conditions that are mediated and/orassociated with Pim kinase proteins. The present invention fulfillsthese needs and offers other related advantages.

BRIEF SUMMARY

The present invention is generally directed to compounds, andpharmaceutical compositions comprising said compounds, where thecompounds have the following general structures (I), (II) and (III):

including stereoisomers, prodrugs, tautomers and pharmaceuticallyacceptable salts thereof, wherein R, R₁, R₂ and X are as defined herein.

These compounds of the present invention have utility over a broad rangeof therapeutic applications, and may be used to treat diseases, such ascancer and various inflammatory conditions, that are mediated at leastin part by protein kinase activity. Accordingly, in one aspect of theinvention, the compounds described herein are formulated aspharmaceutically acceptable compositions for administration to a subjectin need thereof.

In another aspect, the invention provides methods for treating orpreventing a protein kinase-mediated disease, such as cancer, whichmethod comprises administering to a patient in need of such a treatmenta therapeutically effective amount of a compound described herein or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase-mediated disease is a Pimkinase-mediated disease, such as a Pim-1 kinase-expressing cancer.

Another aspect of the invention relates to inhibiting protein kinaseactivity in a biological sample, which method comprises contacting thebiological sample with a compound described herein, or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase is Pim kinase.

Another aspect of this invention relates to a method of inhibitingprotein kinase activity in a patient, which method comprisesadministering to the patient a compound described herein or apharmaceutically acceptable composition comprising said compound. Incertain embodiments, the protein kinase is a Pim kinase.

In another embodiment, the present invention is directed to methods fortreatment of various autoimmune and/or inflammatory conditions mediatedby Pim kinase. The methods comprise administration of any of thedisclosed compounds to a mammal, for example a mammal in need oftreatment for an autoimmune and/or inflammatory condition mediated byPim kinase. Inflammatory conditions which may be treated according tothe disclosed methods include, but are not limited to: osteoarthritis,rheumatoid arthritis, pain, inflammatory bowel diseases, respiratorydisorders, skin disorders or combinations thereof.

These and other aspects of the invention will be apparent upon referenceto the following detailed description and attached figures. To that end,certain patent and other documents are cited herein to more specificallyset forth various aspects of this invention. Each of these documents ishereby incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of a cell-based assay of a representative compound

FIGS. 2A and 2B illustrates cell growth rate in cell culture.

FIGS. 3A and 3B present tumor regression and body weight data.

FIGS. 4A, 4B and 4C show colony growth data.

FIGS. 5A and 5B are graphs showing tumor regression and body weightdata.

FIG. 6 presents data from hematological cancer screening.

FIGS. 7A and 7B show tumor regression and body weight data.

DETAILED DESCRIPTION OF THE INVENTION

According to a general aspect of the present invention, there areprovided compounds useful as protein kinase inhibitors and compositionsand methods relating thereto. Compounds of the invention have structuresset forth in (I), (II) or (III) below:

including stereoisomers, prodrugs, tautomers and pharmaceuticallyacceptable salts thereof, wherein:

X is a direct bond, NH, N(alkyl), S, O, SO or SO₂;

R is H, —OH, halo, alkyl, haloalkyl, alkoxy, haloalkoxy, —NH₂,—NH(alkyl), —N(alkyl)₂, or —CN;

R₁ is optionally substituted carbocycle, optionally substitutedheterocycle or R₁ has the following structure:

where R₁′ is at, each occurrence, independently selected from hydrogencyano, alkyl, alkoxy, halo, haloalkyl, haloalkoxy, —OCF₃, —OCHF₂, —CF₃,—OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ and —N(CH₃)₂ and p is 1, 2 or3.

R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclohexyl, —SO₂—CH₃,—(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-thiophenyl, —(CH₂)_(n)-pyridyl,—(CH₂)_(n)-pyrimidyl, —(CH₂)_(n)-thiomorpholinylsulfone,—(CH₂)_(n)-phenyl, (e.g., unsubstituted phenyl)—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)-morpholinyl,—(CH₂)_(n)OCH₃, —(CH₂)_(n)OH, —(CH₂)_(n)C(CH₃)₂OH or —(CH₂)_(n)N(CH₃)₂,where W is —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹,R¹⁰ and R¹¹ are, at each occurrence, independently H or alkyl; R¹² is—OH, —CN or alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y andz are each independently 0, 1 or 2, and each of the above moieties areoptionally substituted with one or more substituents; or R₂ has one ofthe following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

Unless otherwise stated the following terms used in the specificationand claims have the meanings discussed below:

“Alkyl” refers to a saturated straight or branched hydrocarbon radicalof one to six carbon atoms (i.e., C₁-C₆), preferably one to four carbonatoms, e.g., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl,tert-butyl, pentyl, hexyl, and the like, preferably methyl, ethyl,propyl, or 2-propyl. Representative saturated straight chain alkylsinclude methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and thelike; while saturated branched alkyls include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, and the like. Representative saturatedcyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—CH₂-cyclohexyl, and the like; while unsaturated cyclic alkyls includecyclopentenyl, cyclohexenyl, —CH₂-cyclohexenyl, and the like. Cyclicalkyls are also referred to herein as a “cycloalkyl.” Unsaturated alkylscontain at least one double or triple bond between adjacent carbon atoms(referred to as an “alkenyl” or “alkynyl”, respectively.) Representativestraight chain and branched alkenyls include ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; while representative straight chain and branched alkynyls includeacetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1-butynyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like, preferably methylene, ethylene, or propylene.

“Cycloalkyl” refers to a saturated cyclic hydrocarbon radical of threeto eight carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl.

“Alkoxy” means a radical —OR_(a) where R_(a) is an alkyl as definedabove, e.g., methoxy, ethoxy, propoxy, butoxy and the like.

“Halo” means fluoro, chloro, bromo, or iodo, preferably fluoro andchloro.

“Haloalkyl” means alkyl substituted with one or more, preferably one,two or three, same or different halo atoms, e.g., —CH₂Cl, —CF₃, —CH₂CF₃,—CH₂CCl₃, and the like.

“Haloalkoxy” means a radical —OR_(b) where R_(b) is an haloalkyl asdefined above, e.g., trifluoromethoxy, trichloroethoxy,2,2-dichloropropoxy, and the like.

“Acyl” means a radical —C(O)R_(c) where R_(c) is hydrogen, alkyl, orhaloalkyl as defined herein, e.g., formyl, acetyl, trifluoroacetyl,butanoyl, and the like.

“Aryl” refers to an all-carbon monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of carbon atoms) groups of 6 to12 carbon atoms having a completely conjugated pi-electron system.Examples, without limitation, of aryl groups are phenyl, naphthyl andanthracenyl. The aryl group may be substituted or unsubstituted. Whensubstituted, the aryl group is substituted with one or more substituentsas this term is defined below, more preferably one, two or three, evenmore preferably one or two substituents independently selected from thegroup consisting of alkyl (wherein the alkyl may be optionallysubstituted with one or two substituents), haloalkyl, halo, hydroxy,alkoxy, mercapto, alkylthio, cyano, acyl, nitro, phenoxy, heteroaryl,heteroaryloxy, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino,alkylamino dialkylamino, aryl, heteroaryl, carbocycle or heterocycle(wherein the aryl, heteroaryl, carbocycle or heterocycle may beoptionally substituted).

“Heteroaryl” refers to a monocyclic or fused ring (i.e., rings whichshare an adjacent pair of atoms) group of 5 to 12 ring atoms containingone, two, three or four ring heteroatoms selected from N, O, or S, theremaining ring atoms being C, and, in addition, having a completelyconjugated pi-electron system. Examples, without limitation, ofunsubstituted heteroaryl groups are pyrrole, furan, thiophene,imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,isoquinoline, purine, triazole, tetrazole, triazine, and carbazole. Theheteroaryl group may be substituted or unsubstituted. When substituted,the heteroaryl group is substituted with one or more substituents asthis term is defined below, more preferably one, two or three, even morepreferably one or two substituents independently selected from the groupconsisting of alkyl (wherein the alkyl may be optionally substitutedwith one or two substituents), haloalkyl, halo, hydroxy, alkoxy,mercapto, alkylthio, cyano, acyl, nitro, haloalkyl, haloalkoxy, carboxy,alkoxycarbonyl, amino, alkylamino dialkylamino, aryl, heteroaryl,carbocycle or heterocycle (wherein the aryl, heteroaryl, carbocycle orheterocycle may be optionally substituted).

“Carbocycle” refers to a saturated, unsaturated or aromatic ring systemhaving 3 to 14 ring carbon atoms. The term “carbocycle”, whethersaturated or partially unsaturated, also refers to rings that areoptionally substituted. The term “carbocycle” includes aryl. The term“carbocycle” also includes aliphatic rings that are fused to one or morearomatic or nonaromatic rings, such as in a decahydronaphthyl ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring. The carbocycle group may be substituted orunsubstituted. When substituted, the carbocycle group is substitutedwith one or more substituents as this term is defined below, morepreferably one, two or three, even more preferably one or twosubstituents independently selected from the group consisting of alkyl(wherein the alkyl may be optionally substituted with one or twosubstituents), haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio,cyano, acyl, nitro, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl,amino, alkylamino dialkylamino, aryl, heteroaryl, carbocycle orheterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle maybe optionally substituted).

“Heterocycle” refers to a saturated, unsaturated or aromatic cyclic ringsystem having 3 to 14 ring atoms in which one, two or three ring atomsare heteroatoms selected from N, O, or S(O)_(m) (where m is an integerfrom 0 to 2), the remaining ring atoms being C, where one or two C atomsmay optionally be replaced by a carbonyl group. The term “heterocycle”includes heteroaryl. The heterocyclyl ring may be optionally substitutedindependently with one or more substituents as this term is definedbelow, preferably one, two, or three substituents selected from alkyl(wherein the alkyl may be optionally substituted with one or twosubstituents), haloalkyl, cycloalkylamino, cycloalkylalkyl,cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl, cyanoalkyl, halo,nitro, cyano, hydroxy, alkoxy, amino, alkylamino, dialkylamino,hydroxyalkyl, carboxyalkyl, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, aralkyl, heteroaralkyl, aryl, heteroaryl, carbocycle,heterocycle (wherein the aryl, heteroaryl, carbocycle or heterocycle maybe optionally substituted), aralkyl, heteroaralkyl, saturated orunsaturated heterocycloamino, saturated or unsaturatedheterocycloaminoalkyl, and —COR_(d) (where R_(d) is alkyl). Morespecifically the term heterocyclyl includes, but is not limited to,tetrahydropyranyl, 2,2-dimethyl-1,3-dioxolane, piperidino,N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl,pyrrolidino, morpholino, 4-cyclopropylmethylpiperazino, thiomorpholino,thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide,4-ethyloxycarbonylpiperazino, 3-oxopiperazino, 2-imidazolidone,2-pyrrolidinone, 2-oxohomopiperazino, tetrahydropyrimidin-2-one, and thederivatives thereof. In certain embodiments, the heterocycle group isoptionally substituted with one or two substituents independentlyselected from halo, alkyl, alkyl substituted with carboxy, ester,hydroxy, alkylamino, saturated or unsaturated heterocycloamino,saturated or unsaturated heterocycloaminoalkyl, or dialkylamino.

“Piperidin-2-onyl” refers to a moiety having the following structure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein,

“Thiomorpholinylsulfone” refers to a moiety having the followingstructure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein,

“Tetrahydrothiopyranylsulfone” refers to a moiety having the followingstructure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein,

“Tetrahydrothiopyranyl” refers to a moiety having the followingstructure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein.

“Piperidyl” refers to a moiety having the following structure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein.

Tetrahydropyranyl” refers to a moiety having the following structure:

wherein each R is independently hydrogen, a point of attachment or anoptional substituent as defined herein.

“Tautomer” refers to a compound that results from the formal migrationof a hydrogen atom accompanied by a switch of a single bond and adjacentdouble bond. For example, the enol and keto form of a compound aretautomers of each other.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocyclic group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocycle group is not substituted with the alkyl group.

Lastly, the term “substituted” as used herein means any of the abovegroups (e.g., alkyl, aryl, heteroaryl, carbocycle, heterocycle, etc.)wherein at least one hydrogen atom is replaced with a substituent. Inthe case of an oxo substituent (“═O”) two hydrogen atoms are replaced.“Substituents” within the context of this invention include halogen,hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl,alkoxy, thioalkyl, haloalkyl, hydroxyalkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl, amidynyl(—C(═NH)NH₂), guanidinyl, (—NHC(═NH)NH₂), oximyl (═NOH), —NR_(e)R_(f),—NR_(e)C(═O)R_(f), —NR_(e)C(═O)NR_(e)R_(f),—NR_(e)C(═O)OR_(f)—NR_(e)SO₂R_(f), —OR_(e), —C(═O)R_(e)—C(═O)OR_(e),—C(═O)NR_(e)R_(f), —OC(═O)NR_(e)R_(f), —SH, —SR_(e), —SOR_(B),—S(═O)NH₂, —S(═O)₂R_(e), —OS(═O)₂R_(e), —S(═O)₂OR_(e), wherein R_(e) andR_(f) are the same or different and independently hydrogen, alkyl,haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl or substituted heterocyclealkyl.

Certain illustrative compounds according to structures (I), (II) and(III), for use as described herein, are set forth below.

In some embodiments, the compounds of the present invention havestructure (I). In other embodiments, the compounds have structure (II).In still other embodiments, the compounds have structure (III).

In certain embodiments of structures (I), (II) and (III), X is a directbond. In other embodiments, X is —NH—. In still other embodiments, X is—O—.

In other embodiments of structures (I), (II) and (III), R is H, while indifferent embodiments R is methyl.

In some embodiments of structures (I), (II) and (III), when R₂ is—(CH₂)_(n)-tetrahydropyranyl, then R₁ is not phenyl substituted withcarboxy (—CO₂H).

In some more specific embodiments of structures (I), (II) and (III), R₁is

where R₁′ is at, each occurrence, independently selected from hydrogencyano, alkyl, alkoxy, halo, haloalkyl, haloalkoxy, —OCF₃, —OCHF₂, —CF₃,—OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ and —N(CH₃)₂ and p is 1, 2 or3.

In other embodiments of structures (I), (II) and (III), p is 1. In someembodiments p is 2. In other embodiments, p is 3.

In a more specific aspect of structures (I) and (III) above, R₁ is p,

-   -   and/or m substituted phenyl with one or more occurrences of        cyano, halo, —F, —Cl, —CF₃, —OCF₃, —OCHF₂, —OCH₃, —CH₃, NO₂,        —N(CH₃)₂, —NH₂, —NHSO₂CH₃, —COCH₃, and —OH. In certain other        embodiments, R₁ is p, o and/or m substituted phenyl with one or        more occurrences of cyano, halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃ and        —OH.

In a more specific aspect of structures (I), (II) and (III) above, p is1 and R₁′ is independently selected from hydrogen cyano, halo. —OCF₃,—CF₃, —OCH₃, —OCHF₂, NH₂, NO₂, OH, —COCH₃, —NHSO₂CH₃, and —N(CH₃)₂. Incertain other embodiments of the foregoing, R1′ is cyano, halo, —OCF₃,—OCHF₂, —CF₃, —OCH₃ or —OH, for example in some embodiments R1′ iscyano, chloro, —OCF₃, or —CF₃. In some other embodiments R1′ is cyano.In some other embodiments R1′ is chloro. In some other embodiments R1′is —OCF₃. In some other embodiments R1′ is —CF₃. In certain morespecific embodiments of the foregoing, R1′ is in the meta position.

In a more specific aspect of structures (I), (II) and (III), R₁ has astructure selected from one of the following structures:

In a more specific aspect of structures (I), (II) and (III), R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl (e.g.,unsubstituted phenyl), —(CH₂)_(n)-tetrahydropyranyl,—(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and unless otherwise specified, each of theabove moieties are optionally substituted with one or more substituents;or R₂ is a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In some embodiments, R₂ is —(CH₂)_(n)-unsubstituted phenyl, and in otherembodiments R₂ is —(CH₂)-piperidin-2-only.

In some other embodiments, R₂ is —(CH₂)_(n)-morpholinyl. In even otherembodiments, R2 has one of the following structures:

In certain embodiments, of structure (I) or (II), R1′ is cyano, halo,—OCF₃, —OCHF₂, —CF₃, —OCH₃ or —OH and R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)C(CH₃)₂OH, or astructure selected from one of the following structures:

where W is —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹,R¹⁰ and R¹¹ are, at each occurrence, independently H or alkyl; R¹² is—OH, —CN or alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y andz are each independently 0, 1 or 2 and unless otherwise specified, eachof the above moieties are optionally substituted with one or moresubstituents.

In other certain embodiments of structures (I) and (II) above, R₂ is—(CH₂)_(n)-cyclohexyl

In a more specific aspect of structures (I), (II) and (III) above, R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and unless otherwise specified, each of theabove moieties are optionally substituted with one or more substituents.

In other embodiments of structures (I), (II) and (III) above, R₂ is—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, n is 0,1, 2, 3 or 4; and unless otherwise specified, each of the above moietiesare optionally substituted with one or more substituents

In still other embodiments of structures (I), (II) and (III) above, R₂has a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In still other embodiments of structures (I), (II) and (III) above, R₂has a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In still other embodiments of structures (I), (II) and (III) above, R₂has a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents. In further specificembodiments of structures (I) and (II), X is NH and R₁ is a substitutedor unsubstituted phenyl (where R is as defined above and R₁′ is absentor represents one or more substituents), and the compounds have thefollowing structures (I-A) and (II-A), respectively:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In some specific embodiments of (I-A) and (II-A), R is H. In otherspecific embodiments of (I-A) and (II-A), R is alky, such as methyl, andthe compounds have the following structures (I-Aa) and (II-Aa):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (I-A), (II-A), (I-Aa) and (II-Aa), R₁′is a p, o or m substituent selected from cyano, halo, —OCF₃, —OCHF₂,—CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ and —N(CH₃)₂, and in amore specific embodiment R₁′ is a p, o or m substituent selected fromcyano, —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH. In some embodiments R1′ iscyano. In some embodiments R1′ is chloro. In some embodiments R1′ is—OCF₃. In some embodiments R1′ is —CF₃. In certain more specificembodiments of the foregoing, R1′ is in the meta position.

In some embodiments of (I-A), (II-A), (I-Aa) and (II-Aa), R₁ is selectedfrom one of the following structures:

In more specific embodiments of (I-A), (II-A), (I-Aa) and (II-Aa), R₁has a structure selected from one of the following structures:

In more specific embodiments of structures (I-A), (II-A), (I-Aa) and(II-Aa), R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents.

In some embodiments R₂ is selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-A), (II-A), (I-Aa) and (II-Aa), R₂has a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-A), (II-A), (I-Aa) and (II-Aa), R₂has a structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-A), (II-A), (I-Aa) and (II-Aa), R₂is —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents.

In more specific embodiments of structures (I-A), (II-A), (I-Aa) and(II-Aa), in a more specific embodiment, p is 1 and R₁′ is a p, o or msubstituent selected from cyano, —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH andR₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)C(CH₃)₂OH, or astructure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents, W is —O—, —S(O)_(z)— or>C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ and R¹¹ are, at eachoccurrence, independently H or alkyl; R¹² is —OH, —CN or alkoxy; m is 1,2, 3, 4, 5 or 6; y and z are each independently 0, 1 or 2 and each ofthe above moieties are optionally substituted with one or moresubstituents.

In still further specific embodiments of structures (I) and (II), X is Oand R₁ is a substituted or unsubstituted phenyl (where R is as definedabove and R₁′ is absent or represents one or more substituents), and thecompounds have the following structures (I-B) and (II-B), respectively:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In some specific embodiments of (I-B) and (II-B), R is H. In otherspecific embodiments of (I-B) and (II-B), R is alky, such as methyl, andthe compounds have the following structures (I-Bb) and (II-Bb):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (I-B), (II-B), (I-Bb) and (IIB-b), R₁ issubstituted phenyl having at least one p, o or m substituent selectedfrom cyano, halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃,—NHSO₂CH₃ and —N(CH₃)₂, and in a more specific embodiment R₁ issubstituted phenyl having at least one p, o or m substituent selectedfrom cyano —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH. In some embodiments R1′is cyano. In some embodiments R1′ is chloro. In some embodiments R1′ is—OCF₃. in some embodiments R1′ is —CF₃. In certain more specificembodiments of the foregoing, R1′ is in the meta position.

In some embodiments of (I-B), (II-B), (I-Bb) and (II-Bb), R₁ is selectedfrom one of the following structures:

In more specific embodiments of (I-B), (II-B), (I-Bb) and (II-Bb), R₁has a structure selected from one of the following structures:

In more specific embodiments of structures (I-B), (II-B), (I-Bb) and(II-Bb), R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents. For example, insome embodiments R₂ is selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In other embodiments of structures (I-B), (II-B), (I-Bb) and (II-Bb), R₂is selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-B), (II-B), (I-Bb) and (II-Bb), R₂is —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents.

In more specific embodiments of structures (I-B), (II-B), (I-Bb) and(II-Bb), p is 1, R₁ is a p, o or m substituent selected from cyano,—OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH and R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)n-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)C(CH₃)₂OH, or astructure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents, W is —O—, —S(O)_(z)— or>C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ and R¹¹ are, at eachoccurrence, independently H or alkyl; R¹² is —OH, —CN or alkoxy; m is 1,2, 3, 4, 5 or 6; y and z are each independently 0, 1 or 2 and each ofthe above moieties are optionally substituted with one or moresubstituents.

In yet further specific embodiments of structures (I) and (II), X is S,SO or SO₂ and R₁ is a substituted or unsubstituted phenyl (where R₁′below is absent or represents one or more substituents), and thecompounds have the following structures (I-C) and (II-C), respectively:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In some specific embodiments of (I-A) and (II-A), R is H. In otherspecific embodiments of (I-C) and (II-C), R is alky, such as methyl, andthe compounds have the following structures (I-Cc) and (II-Cc):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (I-C), (II-C), (I-Cc) and (II-Cc), R₁ issubstituted phenyl having at least one p, o or m substituent selectedfrom cyano, halo. —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃,—NHSO₂CH₃ and —N(CH₃)₂, and in an more specific embodiment R₁ issubstituted phenyl having at least one p, o or m substituent selectedfrom cyano, —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH. In some embodiments R1′is cyano. In some embodiments R1′ is chloro. In some embodiments R1′ is—OCF₃. in some embodiments R1′ is —CF₃. In certain more specificembodiments of the foregoing, R1′ is in the meta position.

In other embodiments of (I-C), (II-C), (I-Cc) and (II-Cc) R₁ is selectedfrom one of the following structures:

In more specific embodiments of (I-C), (II-C), (I-Cc) and (II-Cc), R₁has a structure selected from one of the following structures:

In more specific embodiments of (I-C), (II-C), (I-Cc) and (II-Cc), R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents. For example, insome embodiments R₂ is selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-C), (II-C), (I-Cc) and (II-Cc), R₂is selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents.

In other embodiments of structures (I-C), (II-C), (I-Cc) and (II-Cc), R₂is —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents.

In other embodiments of structures (I-B), (II-B), (I-Bb) and (II-Bb), R₂is —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, or —(CH₂)_(n)C(CH₃)₂OH, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents.

In more specific embodiments of structures (I-C), (II-C), (I-Cc) and(II-Cc), p is 1, R₁ is a p, o or m substituent selected from cyano,—OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH and R₂ is

—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-unsubstituted phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)C(CH₃)₂OH, or astructure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents, W is —O—, —S(O)_(z)— or>C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ and R¹¹ are, at eachoccurrence, independently H or alkyl; R¹² is —OH, —CN or alkoxy; m is 1,2, 3, 4, 5 or 6; y and z are each independently 0, 1 or 2 and each ofthe above moieties are optionally substituted with one or moresubstituents.

In still other embodiments, R₂ is

and the present invention is directed to a compound having a structureaccording to structure (I-D) or structure (II-D) below:

or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof,

wherein:

-   -   X is —N(R⁸)— or —O—;    -   W is —O—, —S(O)_(z)— or

-   -   R is H, —OH, halo, alkyl, haloalkyl, alkoxy, haloalkoxy,        —N(R⁸)₂, or —CN;    -   R³, R⁴, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are, at each occurrence,        independently H or alkyl;    -   R⁵ is halo, haloalkyl or haloalkoxy;    -   R⁶ is H, —OH, alkyl or alkoxy;    -   R¹² is —OH, —CN or alkoxy;    -   m is 1, 2, 3, 4, 5 or 6;    -   n is 0, 1, 2, 3 or 4; and    -   y and z are each independently 0, 1 or 2.

In certain embodiments of the foregoing compounds of structure (I-D) or(II-D), R⁶ is H, and in other embodiments R is H or methyl. In stillother embodiments, R⁵ is at the meta position, and the compound has oneof the following structures (I-Da) or (II-Da):

In some embodiments of structures (I-Da) or (II-Da), R⁶ is H, and inother embodiments R is H or methyl.

In some other embodiments, m is 3 or 4, for example is some embodimentsm is 4. In certain embodiments where M is 4, the compound has one of thefollowing structures (IDb) or (IIDb):

In various embodiments of the foregoing compounds of structure (I-D) or(II-D), R⁷ is H. In other various embodiments n is 0 or 1, for examplein some specific embodiments of any of the foregoing n is 0.

In still other various embodiments of the foregoing compounds ofstructure (I-D) or (II-D), at least one of R³ or R⁴ is H, for example incertain embodiments each of R³ and R⁴ is H.

In some embodiments of the foregoing compounds of structure (I-D) or(II-D), R⁵ is —OCF₃, —CF₃, Cl or F. For example, in some of theseembodiments R5 is in the meta position and R⁶ is H.

In still other embodiments of the foregoing compounds of structure (I-D)or (II-D), R¹² is —OH, —CN or —OCH₃. In some embodiments R⁹ is H and inother embodiments R⁹ is methyl. In yet other embodiments, at least oneof R¹⁰ or R¹¹ is methyl, for example in some embodiments each of R¹⁰ andR¹¹ is methyl. In some embodiments y is 0 or 1.

In some other embodiments of the foregoing compounds of structure (I-D)or (II-D), The W is —O—, —S(O)₂—, —CH(OH)—, —CH(CN)—, —C(CH₃)(OH)—,—CH(OCH₃)— or —CH[C(CH₃)₂OH]—. In other embodiments X is —NH—, and insome other embodiments X is —O—.

In still other embodiments of the foregoing compounds of structure (I-D)or (II-D), R₁ is selected from the following structure one of thefollowing structures:

Still other embodiments include compounds of structure (I-D) or (II-D)wherein the compound has a hERG IC₅₀ activity of 10 μM or more, forexample 30 μM or more.

In still other embodiments of structures (I), (II), (I-A), (II-A),(I-B), (II-B), (I-C) or (II-C) above, R₂ is

wherein A represents an optionally substituted 3 to 8-memberedcarbocyclic ring and R³, R⁴, R¹⁰, R¹¹, n and y are as defined above.

In other embodiments of the foregoing, n is 0, and in other embodimentsy is 0 or 1. In some other embodiments, at least one of R³ or R⁴ is H.In some other embodiments, R³ and R⁴ are each H. In some otherembodiments, at least one of R¹⁰ or R¹¹ is methyl. In some otherembodiments, R¹⁰ and R¹¹ are each methyl. In yet other embodiments, A isan optionally substituted 6-membered carbocyclic ring and R² has thefollowing structure:

In some other embodiments of the foregoing, at least one of R¹⁰ or R¹¹is methyl. In some other embodiments, R¹⁰ and R¹¹ are each methyl. Insome embodiments y is 0 and in other embodiments y is 1.

In more specific embodiments R₂ has one of the following structures:

In even other embodiments, R₂ has one of the following structures:

In still other embodiments of structures (I), (II), (I-A), (II-A),(I-B), (II-B), (I-C) or (II-C) above, R₂ is a 3, 4 or 5 memberedcarbocyclic ring and has one of the following structures:

wherein R³, R⁴, R¹⁰, R¹¹, n and y are as defined above.

In other embodiments of the foregoing, n is 0, and in other embodimentsy is 0 or 1. In some other embodiments, at least one of R³ or R⁴ is H.In some other embodiments, R³ and R⁴ are each H. In some otherembodiments, at least one of R¹⁰ or R¹¹ is methyl. In some otherembodiments, R¹⁰ and R¹¹ are each methyl.

In still other embodiments, the present invention is directed to acompound of structure (III), wherein:

X is a direct bond, NH, N(alkyl), S, O, SO or SO₂;

R is H, —OH, —ON, halo, alkyl, haloalkyl, alkoxy or haloalkoxy;

R₁ is carbocycle, substituted carbocycle, heterocycle, or substitutedheterocycle; or a structure selected from:

where R₁′ is a p, o or m substitution with one or more occurrences ofcyano, halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃,—NHSO₂CH₃, —CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂ or —N(CH₃)₂.

R₂ is —(CH₂)_(n)-cyclopropyl, —(CH₂)_(n)-cyclobutyl,—(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl, —SO₂—CH₃,—SO₂—(CH₂)_(n)CH₃, —(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidyl,—(CH₂)-piperidin-2-only, —(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-furyl,—(CH₂)_(n)-thiophene, —(CH₂)_(n)-pyridyl, —(CH₂)_(n)-pyrimidyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)OCH₃, —(CH₂)_(n)OH,—(CH₂)_(n)N(CH₃)₂, —(CH₂)_(n)CH(CH₃)₂OH or —(CH₂)_(n)N(CH₃)₂, where n is0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents; or R₂ is a structure selectedfrom one of the following structures:

where L is optional and, if present, NH, N(alkyl), S, O, SO or SO₂; R₃is one or more optional substituents; and Cycl₁ is a carbocycle,substituted carbocycle, heterocycle or substituted heterocycle; or R₂ isa structure selected from one of the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In a more specific aspect of structure (III) above, R₁ is a 5-6 memberedsaturated, partially unsaturated, or fully unsaturated monocyclic ringhaving 0-3 heteroatoms, where the heteroatoms are selected fromnitrogen, oxygen and sulfur.

In a more specific aspect of structure (III) above, R₁ is p, o or msubstituted phenyl with one or more occurrences of cyano, —F, —Cl, —Br,—CF₃, —OCF₃, —OCH₃, —CH₃, NO₂, —N(CH₃)₂, —NH₂, —NHSO₂CH₃, —NHSO₂CH₂CH₃,—COCH₃, —COOH, —CH₂NH₂, —OH, —SO₂NH₂, —SCH₃, piperazine or morpholine.

In a more specific aspect of structure (III) above, R₁ is an optionallysubstituted pyrazolyl, furyl, thiophene, pyridyl, pyrimidyl, or indolylgroup.

In a more specific aspect of structure (III) above, R₁ has thestructure:

where R₁′ represents one or more optional substituents or, in a morespecific embodiment, is a p, o or m substitution with one or moreoccurrences of cyano, halo, —OCF₃, —CF₃, —OCH₃, —OCHF₂, NH₂, NO₂, OH,—COCH₃, —NHSO₂CH₃, —CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂ or —N(CH₃)₂.

In a more specific aspect of structure (III) above, R₁ has a structureselected from:

In a more specific aspect of structure (III) above, R₂ is 2-butane-1-ol,—SO₂CH₃, —SO₂CH₂CH₃, —CH₂CH₂OCH₃, —CH₂CH₂CH₂OH, or —CH₂CH₂N(CH₃)₂.

In a more specific aspect of structure (III) above, R₂ is optionallysubstituted —(CH₂)_(n)-cyclopropyl, —(CH₂)_(n)-cyclopentyl,—(CH₂)_(n)-cyclohexyl, —(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidyl,—(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-furyl, —(CH₂)_(n)-thiophene,—(CH₂)_(n)-pyridyl, or —(CH₂)_(n)-pyrimidyl.

In a more specific aspect of structure (III) above, R₂ has a structureselected from one of the following structures:

where L is optional and, if present, NH, N(alkyl), S, O, SO or SO₂; R₃is one or more optional substituents; and Cycl₁ is a carbocycle,substituted carbocycle, heterocycle or substituted heterocycle.

In a more specific aspect of structure (III) above, R₂ has the followingstructure:

where L is optional and, if present, NH, N(alkyl), S, O, SO or SO₂; andCycl₁ is a carbocycle, substituted carbocycle, heterocycle orsubstituted heterocycle, and in a more specific embodiment Cycl₁ is a5-6 membered saturated, partially unsaturated, or fully unsaturatedmonocyclic ring having 0-3 heteroatoms, where the heteroatoms areselected from nitrogen, oxygen and sulfur.

In a more specific aspect of structure (III) above, R₂ has a structureselected from:

In a more specific aspect of structure (III) above, R₂ is—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—(CH₂)_(n)-piperidyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)OCH₃ or—(CH₂)_(n)CH(CH₃)₂OH where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents. Forexample, in some embodiments R₂ has a structure selected from one of thefollowing structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In further specific embodiments of structure (III), X is NH and R₁ is asubstituted or unsubstituted phenyl (where R is as defined above and R₁′is absent or represents one or more substituents), and the compoundshave the following structure (III-A):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-A), R is alky, such as methyl, andthe compounds have the following structure (III-Aa):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-A) and (III-Aa), R₁ is substitutedphenyl having at least one p, o or m substituent selected from cyano,halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ and—N(CH₃)₂, and in an more specific embodiment R₁ is substituted phenylhaving at least one p, o or m substituent selected from —OCF₃, —OCHF₂,—CF₃, —OCH₃ and —OH, and in a more specific embodiment R₁ is selectedfrom one of the following structures:

In more specific embodiments of (III-A) and (III-Aa), R₁ has a structureselected from one of the following structures:

In more specific embodiments of (III-A) and (III-Aa), R₂ is—(CH₂)_(1,2)-piperid-4-yl, substituted —(CH₂)_(1,2)-piperid-4-yl,—(CH₂)_(1,2)-piperazin-1-yl, or substituted —(CH₂)_(1,2)-piperazin-1-yl,such as a moiety selected from one of the following structures:

More specifically, in some embodiments R₂ may be selected from one ofthe following structures:

In more specific embodiments of structures (III-A) and (III-Aa), R₂ is—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—(CH₂)_(n)-piperidyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)OCH₃ or—(CH₂)_(n)CH(CH₃)₂OH where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents. Forexample, in some embodiments R₂ has a structure selected from one of thefollowing structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In still further specific embodiments of structure (III), X is O and R₁is a substituted or unsubstituted phenyl (where R is as defined aboveand R₁′ is absent or represents one or more substituents), and thecompounds have the following structure (III-B):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-B), R is alky, such as methyl, andthe compounds have the following structure (III-Bb):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-B) and (III-Bb), R₁ is substitutedphenyl having at least one p, o or m substituent selected from cyano,halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃,—CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂ and —N(CH₃)₂, and in an more specificembodiment R₁ is substituted phenyl having at least one p, o or msubstituent selected from —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH, and in amore specific embodiment R₁ is selected from one of the followingstructures:

In more specific embodiments of (III-B) and (III-Bb), R₁ has a structureselected from one of the following structures:

In more specific embodiments of structures (III-B) and (III-Bb), R₂ is—(CH₂)_(n)-cyclopropyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—SO₂—CH₃, —SO₂—(CH₂)_(n)CH₃, —(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidyl,—(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-furyl, —(CH₂)_(n)-thiophene,—(CH₂)_(n)-pyridyl, —(CH₂)_(n)-pyrimidyl, —(CH₂)_(n)OCH₃, —(CH₂)_(n)OH,or —(CH₂)_(n)N(CH₃)₂, where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents; or astructure selected from one of the following structures:

In more specific embodiments of (III-B) and (III-Bb), R₂ is—(CH₂)_(1,2)-piperid-4-yl, substituted —(CH₂)_(1,2)-piperid-4-yl,—(CH₂)_(1,2)-piperazin-1-yl, or substituted —(CH₂)_(1,2)-piperazin-1-yl,such as moiety selected from one of the following structures:

More specifically, in some embodiments R₂ is selected from one of thefollowing structures:

In more specific embodiments of structures (III-B) and (III-Bb), R₂ is—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—(CH₂)_(n)-piperidyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)OCH₃ or—(CH₂)_(n)CH(CH₃)₂OH where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents. Forexample, in some embodiments R₂ has a structure selected from one of thefollowing structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In yet further specific embodiments of structure (III), X is S, SO orSO₂ and R₁ is a substituted or unsubstituted phenyl (where R₁′ below isabsent or represents one or more substituents), and the compounds havethe following structure (III-C):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-C), R is alky, such as methyl, andthe compounds have the following structure (III-Cc):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In more specific embodiments of (III-C) and (III-Cc), R₁ is substitutedphenyl having at least one p, o or m substituent selected from cyano,halo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃,—CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂ and —N(CH₃)₂, and in an more specificembodiment R₁ is substituted phenyl having at least one p, o or msubstituent selected from —OCF₃, —OCHF₂, —CF₃, —OCH₃ and —OH, and in amore specific embodiment R₁ is selected from one of the followingstructures:

In more specific embodiments of (III-C) and (III-Cc), R₁ has a structureselected from one of the following structures:

In more specific embodiments of structures (III-C) and (iii-Cc), R₂ is—(CH₂)_(n)-cyclopropyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—SO₂—CH₃, —SO₂—(CH₂)_(n)CH₃, —(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidyl,—(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-furyl, —(CH₂)_(n)-thiophene,—(CH₂)_(n)-pyridyl, —(CH₂)_(n)-pyrimidyl, —(CH₂)_(n)OCH₃, —(CH₂)_(n)OH,or —(CH₂)_(n)N(CH₃)₂, where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents; or astructure selected from one of the following structures:

In more specific embodiments of (III-C) and (III-Cc), R₂ is—(CH₂)_(1,2)-piperid-4-yl, substituted —(CH₂)_(1,2)-piperid-4-yl,—(CH₂)_(1,2)-piperazin-1-yl, or substituted —(CH₂)_(1,2)-piperazin-1-yl,such as moiety selected from one of the following structures:

More specifically, in some embodiments R₂ is selected from one of thefollowing structures:

In more specific embodiments of (III-C) and (III-Cc), R₂ is—(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclopentyl, —(CH₂)_(n)-cyclohexyl,—(CH₂)_(n)-piperidyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-thiomorpholinylsulfone, —(CH₂)_(n)-phenyl,—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)OCH₃ or—(CH₂)_(n)CH(CH₃)₂OH where n is 0, 1, 2, 3 or 4 and each of the abovemoieties are optionally substituted with one or more substituents. Forexample, in some embodiments R₂ has a structure selected from one of thefollowing structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.

In still other embodiments of the compound of structure (III), R₂ is

and the present invention is directed to a compound having a structureaccording to structure (III-D) below:

or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof,

wherein:

-   -   X is —N(R⁸)— or —O—;    -   W is —O—, —S(O)_(z)— or

-   -   R is H, —OH, halo, alkyl, haloalkyl, alkoxy, haloalkoxy,        —N(R⁸)₂, or —CN;    -   R³, R⁴, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are, at each occurrence,        independently H or alkyl;    -   R⁵ is halo, haloalkyl or haloalkoxy;    -   R⁶ is H, —OH, alkyl or alkoxy;    -   R¹² is —OH, —CN or alkoxy;    -   m is 1, 2, 3, 4, 5 or 6;    -   n is 0, 1, 2, 3 or 4; and    -   y and z are each independently 0, 1 or 2.

In certain embodiments of the foregoing compounds of structure (III-D)or (II-D), R⁶ is H, and in other embodiments R is H or methyl. In stillother embodiments, R⁵ is at the meta position, and the compound has thefollowing structure (III-Da):

In some embodiments of structure (III-Da), R⁶ is H, and in otherembodiments R is H or methyl.

In some other embodiments, m is 3 or 4, for example is some embodimentsm is 4. In certain embodiments where M is 4, the compound has thefollowing structure (III-Db):

In various embodiments of the foregoing compounds of structure (III-D),R⁷ is H. In other various embodiments n is 0 or 1, for example in somespecific embodiments of any of the foregoing n is 0.

In still other various embodiments of the foregoing compounds ofstructure (III-D), at least one of R³ or R⁴ is H, for example in certainembodiments each of R³ and R⁴ is H.

In some embodiments of the foregoing compounds of structure (III-D), R⁵is —OCF₃, —CF₃, Cl or F. For example, in some of these embodiments R5 isin the meta position and R⁶ is H.

In still other embodiments of the foregoing compounds of structure(III-D), R¹² is —OH, —CN or —OCH₃. In some embodiments R⁹ is H and inother embodiments R⁹ is methyl. In yet other embodiments, at least oneof R¹⁰ or R¹¹ is methyl, for example in some embodiments each of R¹⁰ andR¹¹ is methyl. In some embodiments y is 0 or 1.

In some other embodiments of the foregoing compounds of structure(III-D), The W is —O—, —S(O)₂—, —CH(OH)—, —CH(CN)—, —C(CH₃)(OH)—,—CH(OCH₃)— or —CH[C(CH₃)₂OH]—. In other embodiments X is —NH—, and insome other embodiments X is —O—.

In still other embodiments of the foregoing compounds of structure(III-D), R₁ is selected from one of the following structures:

Still other embodiments include compounds of structure (III-D) whereinthe compound has a hERG IC₅₀ activity of 10 μM or more, for example 30μM or more.

In still other embodiments of structures (III), (III-A), (III-B) or(III-C) above, R₂ is

wherein A represents an optionally substituted 3 to 8-memberedcarbocyclic ring and R³, R⁴, R¹⁰, R¹¹, R¹¹, n and y are as definedabove.

In other embodiments of the foregoing, n is 0, and in other embodimentsy is 0 or 1. In some other embodiments, at least one of R³ or R⁴ is H.In some other embodiments, R³ and R⁴ are each H. In some otherembodiments, at least one of R¹⁰ or R¹¹ is methyl. In some otherembodiments, R¹⁰ and R¹¹ are each methyl. In yet other embodiments, A isan optionally substituted 6-membered carbocyclic ring and R² has thefollowing structure:

In some other embodiments of the foregoing, at least one of R¹° or R¹¹is methyl. In some other embodiments, R¹⁰ and R¹¹ are each methyl. Insome embodiments y is 0 and in other embodiments y is 1.

In more specific embodiments R₂ has one of the following structures:

In even other embodiments, R₂ has one of the following structures:

In still other embodiments of structures (III), (III-A), (III-B) or(III-C) above, R₂ is a 3, 4 or 5 membered carbocyclic ring and has oneof the following structures:

wherein R³, R⁴, R¹⁰, R¹¹, n and y are as defined above.

In other embodiments of the foregoing, n is 0, and in other embodimentsy is 0 or 1. In some other embodiments, at least one of R³ or R⁴ is H.In some other embodiments, R³ and R⁴ are each H. In some otherembodiments, at least one of R¹⁰ or R¹¹ is methyl. In some otherembodiments, R¹⁰ and R¹¹ are each methyl. In more specific aspects ofstructure (I) above, compounds are provided having structures set forthin Table I below (Compounds 8-1 to 8-45 and 8-70 to 8-93).

In more specific aspects of structure (II) above, compounds are providedhaving structures set forth in Table II below (Compounds 8-46 to 8-69and 8-95 to 8-98).

In more specific aspects of structure (III) above, compounds areprovided having structures set forth in Table III below (Compounds 8-99to 8-113).

TABLE I Illustrative Pim inhibitors. EX. Structure 8-1

8-2

8-3

8-4

8-5

8-6

8-7

8-8

8-9

8-10

8-11

8-12

8-13

8-14

8-15

8-16

8-17

8-18

8-19

8-20

8-21

8-22

8-23

8-24

8-25

8-26

8-27

8-28

8-29

8-30

8-31

8-32

8-33

8-34

8-35

8-36

8-37

8-38

8-39

8-40

8-41

8-42

8-43

8-44

8-45

8-68

8-70

8-71

8-72

8-73

8-75

8-76

8-77

8-78

8-79

8-80

8-81

8-82

8-83

8-84

8-85

8-86

8-87

8-88

8-89

8-90

8-91

8-92

8-93

TABLE II Illustrative Pim inhibitors. EX. Structure 8-46

8-47

8-48

8-49

8-50

8-51

8-52

8-53

8-54

8-55

8-56

8-57

8-58

8-59

8-60

8-61

8-62

8-63

8-64

8-65

8-66

8-67

8-69

8-95

8-96

8-97

8-98

TABLE III Illustrative Pim inhibitors. EX. Structure 8-99

8-100

8-101

8-102

8-103

8-104

8-105

8-106

8-107

8-108

8-109

8-110

8-111

8-112

8-113

In certain embodiments, the compound of structure (I-D) or (II-D) hasone of the following structures:

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers”. Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers”. Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog (Cahn, R., Ingold, C., and Prelog, V. Angew. Chem.78:413-47, 1966; Angew. Chem. Internat. Ed. Eng. 5:385-415, 511, 1966),or by the manner in which the molecule rotates the plane of polarizedlight and designated as dextrorotatory or levorotatory (i.e., as (+) or(−)-isomers respectively). A chiral compound can exist as eitherindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of the enantiomers is called a “racemic mixture”.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see discussion in Ch. 4 of ADVANCED ORGANICCHEMISTRY, 4^(th) edition, March, J., John Wiley and Sons, New YorkCity, 1992).

The compounds of the present invention may exhibit the phenomena oftautomerism and structural isomerism. For example, the compoundsdescribed herein may adopt an E or a Z configuration about the doublebond connecting the 2-indolinone moiety to the pyrrole moiety or theymay be a mixture of E and Z. This invention encompasses any tautomericor structural isomeric form and mixtures thereof which possess theability to modulate aurora-2 kinase activity and is not limited to, anyone tautomeric or structural isomeric form.

It is contemplated that a compound of the present invention would bemetabolized by enzymes in the body of the organism such as human beingto generate a metabolite that can modulate the activity of the proteinkinases. Such metabolites are within the scope of the present invention.

Compounds of the present invention can be prepared according to methodsknown to those of ordinary skill in the art, including the methodsexemplified in the examples. In a general sense, compounds of structure(I) can be prepared according to General Reaction Scheme I.

Referring to General Reaction Scheme I, compounds of structure (a),wherein R is as defined above and LG represents an appropriate leavinggroups (e.g., chloro, bromo and the like) can be purchased or preparedaccording to methods known in the art. Reaction of (a) with an activatedaldehyde, such as 2-bromoacetaldehyde, yields fused bycyclic compoundsof structure (b). Activation of an aryl carbon of (b), for example bytreatment with bromine, results in (c), wherein “Act” is an activatinggroup such as bromine. Arylation of (c) with R1-Y, wherein Y is anappropriate activating group) under appropriate conditions (e.g., Suzukicoupling, wherein Y is boronic acid) yields (d). Finally, treatment of(d) with R₂—XH, wherein R2 and X are as defined herein, results incompounds of structure (I). In certain embodiments, a catalyst, such aspalladium (e.g., Buchwald catalysts and the like), may be useful toeffect the coupling of R₂—Z and (d). Optional oxidation or alkylationusing methods known in the art are used to produce compounds havingvarious X moieties.

Compounds of structure (II) can be prepared according to methods knownto those of ordinary skill in the art, including the methods exemplifiedin the examples. A general preparation method for compounds of structure(II) is depicted in General Reaction Scheme II.

In reference to General reaction Scheme II, nitriles of structure (e)can be treated with amino acetals under basic conditions to yield (f).Compounds of structure (f) may then be reacted with hydrazine underacidic conditions (e.g., acetic acid) to yield pyrazoles of structure(g). (g) can then be cyclized by treatment with h in the presence of abase (e.g., sodium ethoxide) to yield (i). Reduction of (i) with anappropriate reagent (e.g., POCl3) yields (j), wherein LG is anappropriate leaving group (e.g., halogen such as chloro or bromo).Finally, compounds of structure (II) are prepared as described for stepD in General Reaction Scheme (I).

Compounds of structure (III) may be prepared according to GeneralReaction Scheme III. Compounds of structure k, wherein LG represents anappropriate leaving group such as bromine, may be purchased fromcommercial sources or prepared according to methods known in the artand/or described herein. Reaction of k with an appropriate halo acid(e.g., acid chloride) results in compounds of structure m. Cyclizationof m by treatment under dehydrating conditions (e.g., acetic acid/HCl)results in compounds of structure n. Finally, compounds of structure(III) are obtained as described for step D in General Reaction Scheme(I).

One skilled in the art will recognize that the above General ReactionSchemes I, II and III may be modified at any number of steps and theorder of steps may be changed. In addition, protecting groups and otherstrategies well known to one of ordinary skill in the art may beemployed, even if not specifically described herein.

The present inventors have discovered that compounds of the presentinvention may be prepared at large scales (e.g., greater than 50 g)according to the general procedures described above. Accordingly, insome specific embodiments, the present invention is directed to a methodfor preparing a compound having the following structure (ID):

or a salt thereof, wherein W is

the method comprising:

a) reacting W—H with a compound of structure (A):

in the presence of CsF; and

b) isolating (ID) as a solid,

wherein W—H indicates a W radical (as defined above) having a hydrogenatom completing its valence.

In certain embodiments of the foregoing method, W—H and (A) are reactedin the presence of a base. For example, in some embodiments the base isDiisopropylethylamine.

In other embodiments, (A) has been prepared by reaction of (B) and (C):

in the presence of a palladium catalyst and a base. For example, thepalladium catalyst may be Pd(PPh₃)₄ and/or the base may be potassiumcarbonate.

In certain other embodiments, the method further comprises treating thesolid with an acid to obtain a salt of (ID). In some embodiments, theacid is HCl, maleic acid or methane sulfonic acid.

Some embodiments of the foregoing method further compriserecrystallizing the solid in a solvent, for example methanol.

In certain specific embodiments, wherein W is

In other embodiments, W is

In still other embodiments, W—H is prepared by a method comprisingalkylation of

or a carboxy or amino protected derivative or a salt thereof. Forexample, alkylation may comprise reaction with a methylmagnesium halidecompound such as methylmagnesium chloride, methylmagnesium bromide ormethylmagnesium iodide.

In even other embodiments, (ID) is prepared at scales of 50 grams orhigher.

A compound of the present invention or a pharmaceutically acceptablesalt thereof, can be administered as such to a human patient or can beadministered in pharmaceutical compositions in which the foregoingmaterials are mixed with suitable carriers or excipient(s). Techniquesfor formulation and administration of drugs may be found, for example,in REMINGTON'S PHARMACOLOGICAL SCIENCES, Mack Publishing Co., Easton,Pa., latest edition.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or pharmaceutically acceptable salts orprodrugs thereof, with other chemical components, such aspharmaceutically acceptable excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

“Pharmaceutically acceptable excipient” refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

“Pharmaceutically acceptable salt” refers to those salts which retainthe biological effectiveness and properties of the parent compound. Suchsalts may include: (1) acid addition salt which is obtained by reactionof the free base of the parent compound with inorganic acids such ashydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid,sulfuric acid, and perchloric acid and the like, or with organic acidssuch as acetic acid, oxalic acid, (D)- or (L)-malic acid, maleic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, tartaric acid, citric acid, succinic acid, glutamic acidor malonic acid and the like, preferably hydrochloric acid or (L)-malicacid; or (2) salts formed when an acidic proton present in the parentcompound either is replaced by a metal ion, e.g., an alkali metal ion,an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base such as ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like.

The compounds of the present invention (I.e., compounds (I), (II) and/or(III) may also act, or be designed to act, as a prodrug. A “prodrug”refers to an agent, which is converted into the parent drug in vivo.Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention, which is, administered as anester (the “prodrug”), phosphate, amide, carbamate or urea.

“Therapeutically effective amount” refers to that amount of the compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. In reference to the treatment ofcancer, a therapeutically effective amount refers to that amount whichhas the effect of: (1) reducing the size of the tumor; (2) inhibitingtumor metastasis; (3) inhibiting tumor growth; and/or (4) relieving oneor more symptoms associated with the cancer.

The term “protein kinase-mediated condition” or “disease”, as usedherein, means any disease or other deleterious condition in which aprotein kinase is known to play a role. The term “proteinkinase-mediated condition” or “disease” also means those diseases orconditions that are alleviated by treatment with a protein kinaseinhibitor. Such conditions include, without limitation, cancers whichexpress Pim kinases, particularly Pim-1 kinase, and otherhyperproliferative disorders associated with Pim kinase expression. Incertain embodiments, the cancer is a cancer of colon, breast, stomach,prostate, pancreas, or ovarian tissue.

The term “Pim kinase-mediated condition” or “disease”, as used herein,means any disease or other deleterious condition in which Pim 1 kinase,Pim 2 Kinase and/or Pim 3 kinase is known to be expressed and/or play arole. The term “Pim kinase-mediated condition” or “disease” also meansthose diseases or conditions that are alleviated by treatment with anPim kinase inhibitor.

As used herein, “administer” or “administration” refers to the deliveryof an inventive compound or of a pharmaceutically acceptable saltthereof or of a pharmaceutical composition containing an inventivecompound or a pharmaceutically acceptable salt thereof of this inventionto an organism for the purpose of prevention or treatment of a proteinkinase-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. In certain embodiments, the preferred routes ofadministration are oral and intravenous.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.In this way, the liposomes may be targeted to and taken up selectivelyby the tumor.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in any conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.Pharmaceutical compositions which may also be used include hard gelatincapsules. The capsules or pills may be packaged into brown glass orplastic bottles to protect the active compound from light. Thecontainers containing the active compound capsule formulation arepreferably stored at controlled room temperature (15-30° C.).

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray using a pressurized pack or a nebulizer and a suitablepropellant, e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD cosolvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD cosolvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This cosolvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a cosolventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the cosolventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the protein kinase-modulating compounds of the invention may beprovided as physiologically acceptable salts wherein the claimedcompound may form the negatively or the positively charged species.Examples of salts in which the compound forms the positively chargedmoiety include, without limitation, quaternary ammonium (definedelsewhere herein), salts such as the hydrochloride, sulfate, carbonate,lactate, tartrate, malate, maleate, succinate wherein the nitrogen atomof the quaternary ammonium group is a nitrogen of the selected compoundof this invention which has reacted with the appropriate acid. Salts inwhich a compound of this invention forms the negatively charged speciesinclude, without limitation, the sodium, potassium, calcium andmagnesium salts formed by the reaction of a carboxylic acid group in thecompound with an appropriate base (e.g. sodium hydroxide (NaOH),potassium hydroxide (KOH), calcium hydroxide (Ca(OH)₂), etc.).

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof protein kinase activity and/or the treatment or prevention of aprotein kinase-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of theprotein kinase activity). Such information can then be used to moreaccurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See, e.g., GOODMAN & GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, Ch. 3, 9^(th) ed., Ed. by Hardman, J., andLimbard, L., McGraw-Hill, New York City, 1996, p. 46.)

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MEGs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

At present, the therapeutically effective amounts of compounds of thepresent invention may range from approximately 2.5 mg/m² to 1500 mg/m²per day. Additional illustrative amounts range from 0.2-1000 mg/qid,2-500 mg/qid, and 20-250 mg/qid.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration, and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

Certain embodiments of the compounds of the present invention are smallmolecule PIM kinase inhibitors that reduce the growth of solid tumorxenografts where the tumorigenicity is mediated by over-expression ofeither PIM-1 or PIM-2, as well as human bladder carcinoma tumors,androgen-independent prostate tumors, and AML models harboring eitherwild-type FLT3 or the FLT3-ITD mutation. While not wishing to be boundby theory, the combination of inhibiting all three PIM family memberstogether in one compound may promote the observed antitumor activity.

Previous work evaluating bladder carcinoma indicated that PIM-1 wasexpressed in over 80% of malignant tissues compared with normalepithelia, and expression was enhanced when comparing specimens derivedfrom bladder cancer patients with invasive versus with non-invasivetumors (Guo, S (2010) J Exp Clin Cancer Res 29:161). Further, it wasshown that shRNA knockdown of PIM-1 in bladder carcinoma cell linesreduced the growth of the cells in vitro, indicating a prominent rolefor PIM-1 in bladder carcinoma (Guo, S (2010) J Exp Clin Cancer Res29:161). As demonstrated in the examples, a representative compound ofthe invention in the bladder cancer model UM-UC-3 in vitro showed aneffective phenocopy by the compound compared to shRNA-mediatedknockdown. The compound reduced the growth of established UM-UC-3xenografts, suggesting that PIM-1 over-expression in patient bladdertumors may be sensitive to perturbation. The examples also demonstratethat PC3 prostate cancer xenografts were inhibited by a representativecompound. While not wishing to be bound by theory, certain embodimentsof the present compounds may advantageously inhibit PIM kinase functionin the solid tumor setting by inhibition of all three PIM kinases for ananti-tumor effect.

AML also represents an important indication of interest for targetingwith PIM kinase inhibitors based on over-expression of PIM-1 and PIM-2(Brault, L (2010) Haematologica 95:1004; Magnuson, N S (2010) FutureOncol 6:1461; Nawijn, M C (2011) Nature Rev Cancer 11:23; Alvarado, Y(2012) Expert. Rev. Hematol 5:81), as well as FLT3 kinase, whereapproximately 25% of patients with AML have tumor cells expressing theFLT3-ITD driver mutation (Kottaridis P D (2001) Blood 98:1752). Inevaluations of AML cell line models for effects of PIM kinaseinhibitors, several groups reported sensitivity in models expressing theFLT3-ITD mutation (Chen, L S (2011) Blood 118:693; Pierre, F (2012) BMCL22:3327; Blanco-Aparicio, C (2011) Cancer Lett 300:145; Fathi, A T(2012) Leuk Res 36:224). PIM-2 over-expression in AML patient specimenssuggests a link between malignancy and expression (Tamburini, J (2009)Blood 114:1618).

As shown in the examples, a representative compound demonstrated a5-fold enhancement in cell cytotoxicity in vitro in the FLT3-ITD mutantcells compared to the wild-type FLT3-expressing cells correlated withxenograft regression versus a partial antitumor effect. Accordingly, itis believed that both populations of AML patients, based on FLT3 status,may benefit from treatment with a PIM kinase inhibitor according to thepresent invention.

As mentioned above, the compounds and compositions of the invention willfind utility in a broad range of diseases and conditions mediated byprotein kinases, including diseases and conditions mediated by PIMkinase. Such diseases may include by way of example and not limitation,cancers such as lung cancer, NSCLC (non small cell lung cancer),oat-cell cancer, bone cancer, pancreatic cancer, skin cancer,dermatofibrosarcoma protuberans, cancer of the head and neck, cutaneousor intraocular melanoma, uterine cancer, ovarian cancer, colo-rectalcancer, cancer of the anal region, stomach cancer, colon cancer, breastcancer, gynecologic tumors (e.g., uterine sarcomas, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina or carcinoma of the vulva), Hodgkin's Disease,hepatocellular cancer, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system (e.g., cancer of the thyroid,pancreas, parathyroid or adrenal glands), sarcomas of soft tissues,cancer of the urethra, cancer of the penis, prostate cancer(particularly hormone-refractory), chronic or acute leukemia, solidtumors of childhood, hypereosinophilia, lymphocytic lymphomas, cancer ofthe bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma,carcinoma of the renal pelvis), pediatric malignancy, neoplasms of thecentral nervous system (e.g., primary CNS lymphoma, spinal axis tumors,medulloblastoma, brain stem gliomas or pituitary adenomas), Barrett'sesophagus (pre-malignant syndrome), neoplastic cutaneous disease,psoriasis, mycoses fungoides, and benign prostatic hypertrophy, diabetesrelated diseases such as diabetic retinopathy, retinal ischemia, andretinal neovascularization, hepatic cirrhosis, angiogenesis,cardiovascular disease such as atherosclerosis, immunological diseasesuch as autoimmune disease and renal disease.

In some embodiments, the compounds and compositions of the invention canbe used in methods for treating cancers such as hematologicalmalignancies. For example, in some embodiments the compounds andcompositions of the invention can be used in methods for treating acutemyeloid leukemia (AML). Other methods include treatment of bladdercancer, or treatment of prostate cancer.

The inventive compounds (i.e., compounds (I), (II) and (III)) can beused in combination with one or more other chemotherapeutic agents. Thedosage of the inventive compounds may be adjusted for any drug-drugreaction. In one embodiment, the chemotherapeutic agent is selected fromthe group consisting of mitotic inhibitors, alkylating agents,anti-metabolites, cell cycle inhibitors, enzymes, topoisomeraseinhibitors such as CAMPTOSAR (irinotecan), biological responsemodifiers, anti-hormones, antiangiogenic agents such as MMP-2, MMP-9 andCOX-2 inhibitors, anti-androgens, platinum coordination complexes(cisplatin, etc.), substituted ureas such as hydroxyurea;methylhydrazine derivatives, e.g., procarbazine; adrenocorticalsuppressants, e.g., mitotane, aminoglutethimide, hormone and hormoneantagonists such as the adrenocorticosteriods (e.g., prednisone),progestins (e.g., hydroxyprogesterone caproate), estrogens (e.g.,diethylstilbesterol), antiestrogens such as tamoxifen, androgens, e.g.,testosterone propionate, and aromatase inhibitors, such as anastrozole,and AROMASIN (exemestane).

Examples of alkylating agents that the above method can be carried outin combination with include, without limitation, fluorouracil (5-FU)alone or in further combination with leukovorin; other pyrimidineanalogs such as UFT, capecitabine, gemcitabine and cytarabine, the alkylsulfonates, e.g., busulfan (used in the treatment of chronicgranulocytic leukemia), improsulfan and piposulfan; aziridines, e.g.,benzodepa, carboquone, meturedepa and uredepa; ethyleneimines andmethylmelamines, e.g., altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolmelamine; and the nitrogen mustards, e.g., chlorambucil (usedin the treatment of chronic lymphocytic leukemia, primarymacroglobulinemia and non-Hodgkin's lymphoma), cyclophosphamide (used inthe treatment of Hodgkin's disease, multiple myeloma, neuroblastoma,breast cancer, ovarian cancer, lung cancer, Wilm's tumor andrhabdomyosarcoma), estramustine, ifosfamide, novembrichin, prednimustineand uracil mustard (used in the treatment of primary thrombocytosis,non-Hodgkin's lymphoma, Hodgkin's disease and ovarian cancer); andtriazines, e.g., dacarbazine (used in the treatment of soft tissuesarcoma).

Examples of antimetabolite chemotherapeutic agents that the above methodcan be carried out in combination with include, without limitation,folic acid analogs, e.g., methotrexate (used in the treatment of acutelymphocytic leukemia, choriocarcinoma, mycosis fungiodes, breast cancer,head and neck cancer and osteogenic sarcoma) and pteropterin; and thepurine analogs such as mercaptopurine and thioguanine which find use inthe treatment of acute granulocytic, acute lymphocytic and chronicgranulocytic leukemias.

Examples of natural product-based chemotherapeutic agents that the abovemethod can be carried out in combination with include, withoutlimitation, the vinca alkaloids, e.g., vinblastine (used in thetreatment of breast and testicular cancer), vincristine and vindesine;the epipodophyllotoxins, e.g., etoposide and teniposide, both of whichare useful in the treatment of testicular cancer and Kaposi's sarcoma;the antibiotic chemotherapeutic agents, e.g., daunorubicin, doxorubicin,epirubicin, mitomycin (used to treat stomach, cervix, colon, breast,bladder and pancreatic cancer), dactinomycin, temozolomide, plicamycin,bleomycin (used in the treatment of skin, esophagus and genitourinarytract cancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

Examples of useful COX-II inhibitors include Vioxx, CELEBREX(celecoxib), valdecoxib, paracoxib, rofecoxib, and Cox 189.

Examples of useful matrix metalloproteinase inhibitors are described inWO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7,1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997),European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29,1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (publishedAug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566(published Jul. 16, 1998), European Patent Publication 606,046(published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21,1999), WO 99/29667 (published Jun. 17, 1999), PCT InternationalApplication No. PCT/IB98/01113 (filed Jul. 21, 1998), European PatentApplication No. 99302232.1 (filed Mar. 25, 1999), Great Britain patentapplication number 9912961.1 (filed Jun. 3, 1999), U.S. Pat. No.5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan.19, 1999), and European Patent Publication 780,386 (published Jun. 25,1997), all of which are incorporated herein in their entireties byreference. Preferred MMP-2 and MMP-9 inhibitors are those that havelittle or no activity inhibiting MMP-1. More preferred are those thatselectively inhibit MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Some specific examples of MMP inhibitors useful in the present inventionare AG-3340, RO 32-3555, RS 13-0830, and compounds selected from:3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methylbenzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[(4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofthese compounds.

Other anti-angiogenesis agents, other COX-II inhibitors and other MMPinhibitors, can also be used in the present invention.

An inventive compound can also be used with other signal transductioninhibitors, such as agents that can inhibit EGFR (epidermal growthfactor receptor) responses, such as EGFR antibodies, EGF antibodies, andmolecules that are EGFR inhibitors; VEGF (vascular endothelial growthfactor) inhibitors; and erbB2 receptor inhibitors, such as organicmolecules or antibodies that bind to the erbB2 receptor, such asHERCEPTIN (Genentech, Inc., South San Francisco, Calif.). EGFRinhibitors are described in, for example in WO 95/19970 (published Jul.27, 1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (publishedJan. 22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998), andsuch substances can be used in the present invention as describedherein.

EGFR-inhibiting agents include, but are not limited to, the monoclonalantibodies C225 and anti-EGFR 22Mab (ImClone Systems, Inc., New York,N.Y.), the compounds ZD-1839 (AstraZeneca), BIBX-1382 (BoehringerIngelheim), MDX-447 (Medarex Inc., Annandale, N.J.), and OLX-103 (Merck& Co., Whitehouse Station, N.J.), and EGF fusion toxin (Seragen Inc.,Hopkinton, Mass.).

These and other EGFR-inhibiting agents can be used in the presentinvention. VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.,South San Francisco, Calif.), can also be combined with an inventivecompound. VEGF inhibitors are described in, for example, WO 01/60814 A3(published Aug. 23, 2001), WO 99/24440 (published May 20, 1999), PCTInternational Application PCT/IB99/00797 (filed May 3, 1999), WO95/21613 (published Aug. 17, 1995), WO 99/61422 (published Dec. 2,1999), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO 01/60814, WO98/50356 (published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar.16, 1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), WO 99/10349 (published Mar. 4, 1999),WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (published Jun. 26,1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438 (published Jan.22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO 98/02437(published Jan. 22, 1998), all of which are incorporated herein in theirentireties by reference. Other examples of some specific VEGF inhibitorsuseful in the present invention are IM862 (Cytran Inc., Kirkland,Wash.); anti-VEGF monoclonal antibody of Genentech, Inc.; and angiozyme,a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron(Emeryville, Calif.). These and other VEGF inhibitors can be used in thepresent invention as described herein. pErbB2 receptor inhibitors, suchas GW-282974 (Glaxo Wellcome plc), and the monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc., The Woodlands, Tex.) and 2B-1 (Chiron),can furthermore be combined with an inventive compound, for example,those indicated in WO 98/02434 (published Jan. 22, 1998), WO 99/35146(published Jul. 15, 1999), WO 99/35132 (published Jul. 15, 1999), WO98/02437 (published Jan. 22, 1998), WO 97/13760 (published Apr. 17,1997), WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No. 5,587,458(issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issued Mar. 2,1999), which are all hereby incorporated herein in their entireties byreference. ErbB2 receptor inhibitors useful in the present invention arealso described in U.S. Pat. No. 6,284,764 (issued Sep. 4, 2001),incorporated in its entirety herein by reference. The erbB2 receptorinhibitor compounds and substance described in the aforementioned PCTapplications, U.S. patents, and U.S. provisional applications, as wellas other compounds and substances that inhibit the erbB2 receptor, canbe used with an inventive compound, in accordance with the presentinvention.

An inventive compound can also be used with other agents useful intreating cancer, including, but not limited to, agents capable ofenhancing antitumor immune responses, such as CTLA4 (cytotoxiclymphocyte antigen 4) antibodies, and other agents capable of blockingCTLA4; and anti-proliferative agents such as other farnesyl proteintransferase inhibitors, for example the farnesyl protein transferaseinhibitors described in the references cited in the “Background”section, of U.S. Pat. No. 6,258,824 B1.

The above method can also be carried out in combination with radiationtherapy, wherein the amount of an inventive compound in combination withthe radiation therapy is effective in treating the above diseases.

Techniques for administering radiation therapy are known in the art, andthese techniques can be used in the combination therapy describedherein. The administration of the compound of the invention in thiscombination therapy can be determined as described herein.

Pim kinases also play a role in immune regulation and inflammatorystates. Accordingly, one embodiment of the invention is directed tomethods for treatment of an autoimmune or inflammatory disorder, diseaseor condition mediated by Pim kinase. The disclosed methods compriseadministering an effective amount of any of the disclosed compounds to amammal, for example a mammal in need of treatment for an autoimmune orinflammatory disease, disorder or condition. Inflammatory diseases,disorders or conditions treatable according to the disclosed methodsinclude, but are not limited to: osteoarthritis, rheumatoid arthritis,pain, inflammatory bowel diseases, respiratory disorders, skin disordersor combinations thereof.

In some embodiments, the inflammatory bowel diseases are selected fromCrohn's disease, ulcerative colitis, irritable bowel syndrome andcombinations thereof.

In some other embodiments, the respiratory disorders are selected fromasthma, rhinitis, chronic obstructive pulmonary disease, bronchitis,nasal polyposis, nasal congestion, farmer's lung, fibroid lung, coughand combinations thereof.

In yet other embodiments, the skin disorders are selected fromdermatitis, cutaneous eosinophilias, Lichen planus, urticaria,psoriasis, pruritus, angiodermas, corneal ulcers, chronic skin ulcers,conjunctivitis, vasculitides, uveitis, erythemas and combinationsthereof.

While the compounds and compositions of the invention find utility in abroad range of diseases and conditions, including those described above,some embodiments include compounds which have reduced side effectscompared to other known therapeutics. One such side effect may includehERG inhibition. hERG (the human Ether-à-go-go-Related Gene) is a genethat encodes a protein which contributes to the electrical activity ofthe heart. Some clinically successful drug candidates have the tendencyto inhibit hERG which may compromise the electrical activity of theheart and can result in a potentially fatal disorder called long QTsyndrome. Thus, drugs capable of treating any of the diseases,conditions or disorders described herein, without substantialconcomitant inhibition of hERG, may be advantageous in certain methodsof treatment. Accordingly, certain embodiments of the present inventioninclude compounds which have a hERG inhibition IC₅₀ of greater than 1μM, greater than 10 μM, greater than 30 μM or even greater than 50 μM(as determined by the Fast Patch assay available from WuXiApptec(Shanghai China)). In further embodiments of the foregoing, the compoundis also effective for treatment of the Pim kinase mediated diseasesdescribed above.

The invention will be further understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES

Representative compounds of the present invention were synthesizedaccording the synthetic procedures detailed herein.

All solvents and reagents were available from Aldrich or VWR chemicalsand used as supplied or purified by standard laboratory methods asrequired. NMR spectra were recorded on a Varian Unity at 400 MHz (¹H) at25° C. Chemical shifts are reported in ppm and referenced internally toresidual CHCl₃ for (d 7.26) or CH₃OH for (d 3.33). Low resolution massspectrometry was performed by The Mass Spectrometry and Proteomics Corefacility at the University of Utah. Flash chromatography was performedon Combifalsh (Yamazen) with normal phase silica gel column (RediSep)and CH₂Cl₂/CH₃OH solvent system. TLC used pre-coated silica gel aluminumsheets.

Antibodies and Reagents

PIM-1, PIM-2 and actin antibodies were purchased from Cell SignalingTechnology (Danvers, Mass.) (Cat #3247, 4730 and 3700). IRDye 800CW goatanti-rabbit and IRDye 680 goat anti-mouse secondary antibodies werepurchased from LI-COR (Lincoln, Nebr.). HEK-293, NIH-3T3, 22RV-1,UM-UC-3, PC-3 MV-4-11, GDM-1, RPMI-8226, NCI-H929, Pfeiffer, SU-DHL-6,Toledo, Z-138, Jeko-1, Maver-1, Mino, MC116, JM-1, SupT1, U937 and K562cell lines were purchased from ATCC (Manassas, Va.), while the MOLM-13,ML-2 and PL-21 cell lines were purchased from DSMZ (Braunschweig,Germany), and the 22RV1/PIM-1 over-expression cell line was licensedfrom the lab of Anjali Jain, Ph.D. (Cedars Sinai, Calif.). TheNIH-3T3/PIM-2 over-expression cell lines were generated at Epoch LifeScience (Missouri City, Tex.) by transfecting the NIH-3T3 cell line witha PIM-2 over-expression plasmid encoding a neomycin resistance gene andselecting a stable pool resistant to G418. PIM-1, PIM-2 and BAD plasmidswere purchased from Origene (Rockville, Md.). Human shRNA lentiviralparticles were purchased from the Sigma-Aldrich MISSION collection (St.Louis, Mo.). For cell culture, RPMI 1640 and EMEM media with glutaminewere supplemented with 10% fetal bovine serum and 1×Penicillin-Streptomycin (Life Technologies, Grand Island N.Y.). ATPlitewas purchased from Perkin Elmer (Shelton, Conn.).

Cell-Based Assays

HEK-293 cells (1×10⁴) were seeded in a 96-well plate using EMEM mediawith serum and incubated overnight at 37° C. in 5% CO₂. Adherent cellswere transfected the next day using Effectene (Qiagen) with 0.1 μgBAD+300 pg of PIM-1 or PIM-1 KD (K67M) overexpression plasmids for 18hours. Cells were serum starved for an additional 18 hours, then treated2 hours with various concentrations of SGI-9481 or 0.5% carrier DMSOcontrol. Media was removed and cells were lysed using 25 μl 1× lysisbuffer from the AlphaScreen SureFire Phospho-BAD (Ser112) kit fromPerkin Elmer. Lysates (4 μl) were transferred to a 384-well proxiplate,and 5 μl of acceptor mix was added to each well and incubated for 2hours at room temperature. Donor mix (2 μl) was added to each well, andsamples incubated for 2 hours at room temperature and reactions measuredon an Envision plate reader (Perkin Elmer) with AlphaScreencapabilities. Non-specific phosphorylation of BAD was subtracted usingvalues from PIM-1 KD transfected cells, and the remaining wells werenormalized to the DMSO control wells. EC₅₀ values were calculated usingGraphPad Prism software (La Jolla, Calif.).

Western Blot Analysis

Protein lysates were prepared from cells using an NP-40 based cell lysisbuffer (30 mM Tris-HCl pH 7.4, 120 mM NaCl, 1% NP-40, 1× proteaseinhibitor cocktail, 1× phosphatase inhibitor cocktail I and II (Sigma)).Cells were incubated with lysis buffer for 20 minutes on ice,centrifuged at 13000×g for 5 min at 4° C., and clarified lysate wasquantified using the BCA protein assay kit (Thermo Scientific, Rockford,Ill.). Protein (10 μg) from PIM-1 and PIM-2 overexpressing cells andmatching parental controls or 50 μg of protein from UM-UC-3 cells 48hours post-transduction were loaded onto a 4-12% Bis-Tris NuPAGE Novexgel, transferred to nitrocellulose membrane using an iBlot transfersystem. Membranes were probed with antibodies against PIM-1, PIM-2 oractin, then probed with rabbit or mouse IRDye secondary antibodies andimaged on an Odyssey fluorescence imaging system (LI-COR).

shRNA Transduction

UM-UC-3 cells (2.5×10⁵) were seeded in a 6-well plate in complete RPMI1640 media, and allowed to adhere overnight at 37° C. in 5% CO₂. Cellswere transduced with 8 μg/mL polybrene (Sigma-Aldrich) and Lentiviralparticles at an MOI of 50 based on titer values pre-determined bySigma-Aldrich using a p24 ELISA for each batch of shRNA. Followingovernight transduction, viral particle containing media was removed andreplaced with fresh complete media, and cells were cultured for anadditional 48 hours at 37° C. in 5% CO₂. Cells were trypsinized andfractions of the transduced cells were collected for colony formationgrowth assays, while the remaining cells were collected for RNA andprotein isolation.

RT-PCR

RNA from 0.5×10⁶ cells was isolated on a QIAcube (Qiagen, Santa Clarita,Calif.) using the protocol for purification of total RNA from animalcells (QIAshredder homogenization and on-column DNase digest), andquantified using a Nanodrop 8000 spectrophotometer (Thermo Electron,West Palm Beach, Fla.). Total RNA (1 μg) was converted to cDNA in a 20μl reaction using the iScript cDNA synthesis kit (Bio-Rad, Hercules,Calif.) by incubating the reaction components for 5 minutes at 25° C.,30 minutes at 42° C., followed by 5 minutes at 85° C. The cDNA reaction(2 μl) was used in a 20 μl PCR multiplex reaction using 1×FAM-labeledPIM-1, VIC-labeled actin Taqman primer sets and the Taqman geneexpression master mix from Life Technologies on an iQ5 Real-Time PCRmachine (Bio-Rad). An 8-point, half-log standard curve was generated forPIM-1 and actin messages using RNA from untreated cells. A lineartrendline (with an R squared value >0.99) was generated by plotting logconcentrations of standard vs. Ct values generated from the real-timePCR reactions. Relative message levels from shRNA treated samples werecalculated based on the standard curve, normalized to actin and comparedto the non-target shRNA control.

Colony Formation Assay

For shRNA growth experiments, 500 UM-UC-3 cells were seeded in a 12-wellplate 48 hours post-transduction and cultured for 8-10 days at 37° C. in5% CO₂. Cells were fixed with 4% paraformaldehyde in PBS, washed twicewith PBS, and stained with a crystal violet solution (1% crystal violet,10% ethanol in water). Stained cells were washed thrice with water, andimaged after drying on a GelCount colony counter (Oxford Optronix Ltd.,Oxford, UK). Total staining intensity per well was determined by lysisof cells with 200 μl of Triton X-100 lysis buffer (1% Triton X-100, 50mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA). Lysates (100 μl) from eachwell were transferred to a clear 96-well plate and absorbance at 560 nmwas determined on an Envision microplate reader. IC₅₀ values weredetermined using GraphPad Prism software. For compound treated PC-3 andUM-UC-3, cells were seeded and stained as above, but were incubated withvarious concentrations of test compound or DMSO one day after seeding.

Proliferation Assay

Adherent or suspension cancer cell lines were seeded at 2000-5000cells/well in a 96-well plate, or 1000-2000 cells/well in a 384-wellplate. Suspension cells were treated with drug the same day as seeding,while adherent cells were incubated overnight at 37° C. in 5% CO₂ priorto drug treatment. All cells were treated for 72 hours, then lysed withATPlite (Perkin Elmer) and measured on an Envision microplate reader.IC₅₀ values were determined using GraphPad Prism software.

Tumor Xenograft Studies

Male and female Nu/Nu mice were purchased from Harlan Sprague Dawley(Indianapolis, Ind.). Female Nu/Nu mice were used for all xenograftevaluations with the exception of the PC-3 xenografts where male Nu/Numice were used. Cell lines were expanded in vitro in complete media, andif adherent were harvested by trypsin-EDTA, centrifuged and resuspendedin PBS 1:1 with Matrigel (BD Bioscienes). Cells were inoculatedsubcutaneously in the right hind flank of mice. When tumors reached100-200 mm³ by caliper measurement, mice were randomized and dosing ofSGI-9481 or vehicle control began and continued every day for 5 days(QD×5) with 2 days off for 18-21 days. Tumor volumes and body weightswere determined twice a week, and tumor weights were measured at thecompletion of the translational xenograft studies.

Statistical Analyses

All statistical analyses were performed by parametric ANOVA test.

Example 1 Synthesis of Illustrative Pim-1 Kinase Inhibitors 1.4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexanol(EX. 8-1)

3-bromo-6-chloroimidazo[1,2-b]pyridazine was prepared according toprocedures described in U.S. Pat. No. 7,750,007, which is herebyincorporated by reference in its entirety. To a solution of3-bromo-6-chloroimidazo[1,2-b]pyridazine (5 g, 21.5 mmol) and3-(trifluoromethoxy)phenylboronic acid (4.43 g, 21.5 mmol) indioxane/H₂O (100 mL, 4:1) was added K₂CO₃ (6.8 g, 64.5 mmol) andPd(PPh₃)₄ (1.9 g, 2.58 mmol), the mixture was stirred at 110° C. for 3h. The solution was concentrated, partitioned in EtOAc/H₂O. The aqueouslayer was washed with EtOAc (50 mL) for 3 times. The collected organiclayers were dried over Na₂SO₄, concentrated and purified by columnchromatography to give compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (3.2 g,with 63% purity, yield 30%) as a brown solid.

To a solution of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (200 mg,0.64 mmol) and trans-4-aminocyclohexanol hydrogenchloride (184 mg, 1.59mmol) in NMP (2.0 mL) was added NaHCO₃ (161 mg, 1.91 mmol), the mixturewas stirred at 180° C. for 45 mins under microwave irridation. Themixture was purified by flash chromatograph to give4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanol(50 mg, 0.127 mmol, 16%) as a pale yellow solid.

¹H-NMR (DMSO-d₆/400 MHz): δ 8.49 (s, 1H), 8.34 (brs, 1H), 8.07 (d, J=8Hz, 1H), 7.99 (d, J=10 Hz, 1H), 7.67 (m, 2H), 7.45 (d, J=7.6 Hz, 1H),7.12 (m, J=10 Hz, 1H), 3.54 (s, 2H), 2.05 (d, J=9.6 Hz, 2H), 1.87 (d,J=9.6 Hz, 2H), 1.25 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 393.4.

2.4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-cis-cyclohexanol(EX. 8-2)

To a solution of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (200 mg,0.64 mmol) and cis-4-aminocyclohexanol hydrogenchloride (367 mg, 3.19mmol) in NMP (2.0 mL) was added NaHCO₃ (268 mg, 3.19 mmol), the mixturewas stirred at 180° C. for 45 mins under microwave irridation. Themixture was purified by flash chromatograph to give4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanol(80 mg, 32%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.84 (s,1H), 7.58 (d, J=8.0 Hz, 1H), 7.51 (m, 1H), 7.21 (d, J=8.4 Hz, 1H), 6.75(d, J=9.6 Hz, 1H), 3.88 (m, 2H), 1.79 (m, 8H). MS (ES⁺, m/z): (M+H)⁺:393.4.

3.2-Methyl-4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)butan-2-ol(EX. 8-3)

To a solution of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (300 mg,0.96 mmol) and 4-amino-2-methylbutan-2-ol hydrogenchloride (493 mg, 4.78mmol) in NMP (2.0 mL) was added NaHCO₃ (516 mg, 4.78 mmol), the mixturewas stirred at 180° C. for 45 mins under microwave irridation. Themixture was purified by flash chromatograph to give2-methyl-4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)butan-2-ol(175 mg, 48%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 9.30 (s, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.83 (s,1H), 7.61 (d, J=10.0 Hz, 1H), 7.51 (t, J=8.4 Hz, 1H), 7.20 (d, J=8.0 Hz,1H), 6.71 (d, J=10.0 Hz, 1H), 3.88 (t, J=7.6 Hz, 1H), 1.89 (t, J=7.6 Hz,1H), 1.26 (s, 6H). MS (ES⁺, m/z): (M+H)⁺: 381.5.

4.4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)oxy)-trans-cyclohexanol(EX. 8-4)

A solution of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (0.3 g,0.96 mmol) and cyclohexanediol (0.11 g, 0.96 mmol) in toluene 5 mL wasadded potassium tertiary butoxide (0.165 g, 1.72 mmol), rac-BINAP (36mg, 0.057 mmol) and Pd₂(dba)₃ (26 mg, 0.029 mmol) and the mixture washeated at 120° C. for 4 h under microwave irridation. The resulting darkbrown solution was cooled down and was concentrated under reducedpressure. The solid was further purified by using combiflashchromatography (12 g column), eluent: 0-10% methanol/DCM and obtainedproduct4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)oxy)cyclohexanol(50 mg, 0.127 mmol, 13% yield).

¹H-NMR (CD₃OD/400 MHz): δ 8.12 (s, 1H), 7.93 (s, 1H), 7.89 (m, 1H), 7.87(s, 1H), 7.87 (m, 1H), 7.54 (t, J=8.0 Hz, 1H), 7.25 (d, J=9.2 Hz, 1H),6.85 (d, J=9.6 Hz, 1H), 4.98 (m, 1H), 3.69 (m, 1H), 2.22 (m, 2H), 2.02(m, 2H), 1.62 (m, 2H), 1.45 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 394.5.

5.(1R,3R)3-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclopentanol(EX. 8-5)

EX. 8-5 was prepared by similar procedures as in EX. 8-1 usingtrans-3-aminocyclopentanol.

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 7.98 (d, J=8.8 Hz, 1H), 7.80 (s,1H), 7.58 (d, J=8.0 Hz, 1H), 7.50 (m, 1H), 7.19 (m, 1H), 6.72 (d, J=8.0Hz, 1H), 4.28 (m, 1H), 4.21 (m, 1H), 2.43 (m, 1H), 2.15 (m, 1H), 1.83(m, 3H), 1.58 (m, 1H). MS (ES⁺, m/z): (M+H)⁺: 379.5.

6.2-Methyl-1-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)propan-2-ol(EX. 8-6)

To the solution of compound 1-amino-2-methylpropan-2-ol (55 mg, 0.57mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (150 mg,0.48 mmol) in 3 mL DMSO was added DIEA (0.21 mL, 0.96 mmol) and 10 mgCsF, the solvent was stirred for 5 h at 120° C., Then the mixture waspurified by HPLC to afford the compound2-methyl-1-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)propan-2-ol(25 mg, 12%) as a white solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.28-8.24 (d, J=15.2 Hz, 2H), 7.99-7.92 (m,2H), 7.66-7.62 (t, J=8.4 Hz, 1H), 7.41-7.39 (m, 1H), 7.34-7.31 (d, J=9.6Hz, 1H), 3.42 (d, 2H), 1.28-1.27 (d, J=6 Hz, 6H). MS (ES⁺, m/z): (M+H)⁺:367.4.

7.3-(((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)-trans-cyclobutanol(EX. 8-7)

EX. 8-5 was prepared by similar procedures as in EX. 8-1 usingtrans-3-(aminomethyl)cyclobutanol.

¹H-NMR (CD₃OD/400 MHz): δ 8.43 (s, 1H), 7.94 (d, J=8.8 Hz, 1H), 7.83 (s,1H), 7.59 (d, J=8.0 Hz, 1H), 7.48 (m, 1H), 7.19 (m, 1H), 6.70 (d, J=8.0Hz, 1H), 4.38 (m, 1H), 3.40 (m, 1H), 2.57 (m, 1H), 2.10 (m, 5H). MS(ES⁺, m/z): (M+H)⁺: 379.5.

8.(3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-trans-1,4-diamine(EX. 8-8)

A solution of pyridazine (0.3 g, 0.96 mmol) and1,4-trans-cyclohexanediamine (0.11 g, 0.96 mmol) in toluene 5 mL wasadded potassium tertiary butoxide (0.165 g, 1.72 mmol), rac-BINAP (36mg, 0.057 mmol) and Pd₂(dba)₃ (26 mg, 0.029 mmol) and the mixture washeated at 100° C. for 16 h under microwave irridation. The resultingdark brown solution was cooled down and was concentrated under reducedpressure. The solid was further purified by using combiflashchromatography (12 g column), eluent: 0-10% methanol/DCM and obtainedproduct4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)oxy)cyclohexanol(170 mg, 0.434 mmol, 45% yield.).

¹H-NMR (CD₃OD/400 MHz): δ 8.30 (s, 1H), 8.00 (m, 1H), 7.83 (s, 1H), 7.60(d, J=9.2 Hz, 1H), 7.50 (t, J=8.0 Hz, 1H), 7.21 (d, J=9.2 Hz, 1H), 6.68(d, J=9.6 Hz, 1H), 3.68 (m, 1H), 2.70 (m, 1H), 2.24 (m, 2H), 1.98 (m,2H), 1.33 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 375.4.

9.1-Methyl-4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexanol(EX. 8-9)

EX. 8-9 was prepared by similar procedures as in EX. 8-1 usingtrans-4-amino-1-methylcyclohexanol.

¹H-NMR (CD₃OD/400 MHz): δ 8.23 (s, 1H), 7.93 (d, J=8.8 Hz, 1H), 7.76 (s,1H), 7.54 (d, J=8.0 Hz, 1H), 7.42 (m, 1H), 7.16 (m, 1H), 6.60 (d, J=8.0Hz, 1H), 3.77 (m, 1H), 2.09 (m, 2H), 1.72 (m, 2H), 1.45 (m, 4H), 1.30(s, 3H). MS (ES⁺, m/z): (M+H)⁺: 407.5.

10.4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-10)

EX. 8-10 was prepared by similar procedures as in EX. 8-1 using2-(trans-4-aminocyclohexyl)propan-2-ol.

¹H-NMR (CD₃OD/400 MHz): δ 8.45 (s, 1H), 7.95 (d, J=8.0 Hz, 1H), 7.82 (s,1H), 7.59 (d, J=8.0 Hz, 1H), 7.50 (t, J=8.0 Hz, 1H), 7.21 (d, J=7.6 Hz,1H), 6.68 (d, J=10.0 Hz, 1H), 3.50 (m, 1H), 2.09 (m, 2H), 2.27 (m, 2H),1.95 (m, 2H), 1.40 (m, 1H), 1.26 (m, 2H), 1.14 (s, 6H). MS (ES⁺, m/z):(M+H)⁺: 435.6.

11.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-11)

EX. 8-11 was prepared by similar procedures as in EX. 8-1 usingtrans-4-methoxycyclohexanamine.

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.87 (s,1H), 7.63 (d, J=8.0 Hz, 1H), 7.54 (s, 1H), 7.26 (s, 1H), 6.72 (d, J=10.0Hz, 1H), 3.76 (m, 1H), 3.39 (s, 6H), 3.30 (m, 1H), 2.27 (m, 2H), 2.15(m, 2H), 1.40 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 407.6.

12.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-12)

EX. 8-12 was prepared by similar procedures as in EX. 8-1 usingtetrahydro-2H-pyran-4-amine.

¹H-NMR (CD₃OD/400 MHz): δ 8.37 (s, 1H), 7.95 (d, J=8.0 Hz, 1H), 7.87 (s,1H), 7.63 (d, J=8.0 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.23 (m, 1H), 6.72(d, J=10.0 Hz, 1H), 3.98 (m, 5H), 2.11 (m, 2H), 1.59 (m, 2H). MS (ES⁺,m/z): (M+H)⁺: 379.5.

13.4-(((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-13)

A mixture of compound dihydro-2H-thiopyran-4(3H)-one (0.75 g, 6.46 mmol)and TosMIC (0.138 g, 7.11 mmol) in DME (25 mL) was cooled to 0° C. and asolution of potassium tert-butoxide (0.145 g, 12.9 mmol) in tert-butanol(13 mL) added dropwise. The mixture was then allowed to warm to roomtemperature and stirred for 3 h before dilution with DCM, washing withSat. sodium bicarbonate and dried over Na₂SO₄. Removal of the solvent invacuo to afford the compound tetrahydro-2H-thiopyran-4-carbonitrile (1g, crude) as pale brown oil.

¹H-NMR (CDCl₃/400 MHz): δ 7.98-7.94 (m, 1H), 7.44-7.43 (d, J=1.6 Hz,1H), 7.37-7.34 (m, 1H).

LiAlH₄ (45 mg, 11.8 mmol) in 15 mL THF was cooled to 0° C. under N₂. Theaddition funnel was charged with 10 mL compoundtetrahydro-2H-thiopyran-4-carbonitrile (1 g, 8 mmol) in 5 mL THF, therate of addition was set to maintain the temperature below 10° C. Thereaction mixture was allowed to gradually warm to room temperature andstirred for overnight. The reaction mixture was then cooled to about 5°C. and Na₂SO₄.10H₂O was added in portions to maintain the temperature atabout 10° C. The reaction mixture was filtered and the salts were washedwith warm THF. The filtrate was combined and concentrated to afford thecompound (tetrahydro-2H-thiopyran-4-yl)methanamine (150 mg, crude) asyellow oil.

To the solution of compound (tetrahydro-2H-thiopyran-4-yl)methanamine(150 mg, 1.14 mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (358 mg,1.14 mmol) in 3 mL DMSO was added DIEA (0.22 mL, 2.28 mmol) and 10 mgCsF, the solvent was stirred for 5 h at 120° C., Then the mixture waspurified by HPLC to afford the compoundN-((tetrahydro-2H-thiopyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(26 mg, 30%) as yellow oil.

¹H-NMR (CDCl₃/400 MHz): δ 8.29 (s, 1H), 8.23 (s, 1H), 7.98-7.95 (m, 2H),7.64-7.60 (t, J=8 Hz, 1H), 7.41-7.39 (d, J=8 Hz, 1H), 7.25-7.23 (d, J=10Hz, 1H), 3.24-3.22 (d, J=6.8 Hz, 2H), 2.68-2.55 (m, 4H), 2.14-2.10 (m,2H), 1.80-1.78 (t, J=3.2 Hz, 1H), 1.44-1.37 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 409.5.

The compoundN-((tetrahydro-2H-thiopyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(70 mg, 0.171 mmol) was dissolved in CH₃OH (10 mL). A solution of(NH₄)Mo₇O₂₄ (84 mg, 0.07 mmol) in H₂O₂ (290 mg, 2.56 mmol) was added tothe mixture dropwise. After stirred for 30 min, the solvent was removedand purified by column to afford the compound4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (23 mg, 30%) as a yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.20 (s, 1H), 7.90-7.87 (m, 1H), 7.75 (s, 1H),7.56-7.53 (d, J=9.6 Hz, 1H), 7.44-7.40 (t, J=8.4 Hz, 1H), 7.14-7.12 (m,1H), 6.65-6.62 (d, J=9.6 Hz, 1H), 3.25-3.20 (m, 2H), 3.02-2.95 (m, 4H),2.15-2.11 (d, J=12.8 Hz, 2H), 2.08-1.95 (m, 1H), 1.81-1.69 (m, 2H). MS(ES⁺, m/z): (M+H)⁺: 441.5.

14.4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-14)

EX. 8-14 was prepared by similar procedures as in EX. 8-1 using4-aminotetrahydro-2H-thiopyran 1,1-dioxide.

¹H-NMR (CD₃OD/400 MHz): δ 8.20 (s, 1H), 8.01 (d, J=7.6 Hz, 1H), 7.82 (s,1H), 7.67 (d, J=7.6 Hz, 1H), 7.55 (t, J=8.0 Hz, 1H), 7.23 (d, J=8.0 Hz,1H), 6.75 (d, J=10.0 Hz, 1H), 4.01 (m, 1H), 3.20 (m, 4H), 2.48 (m, 2H),2.24 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 441.5.

15.4-(3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)thiomorpholine1,1-dioxide (EX. 8-15)

EX. 8-15 was prepared by similar procedures as in EX. 8-1 usingthiomorpholine 1,1-dioxide.

¹H-NMR (CD₃OD/400 MHz): δ 8.10 (s, 1H), 7.98 (m, 2H), 7.89 (d, J=7.6 Hz,1H), 7.57 (t, J=8.0 Hz, 1H), 7.31 (d, J=10.0 Hz, 1H), 7.26 (d, J=8.4 Hz,1H), 4.18 (t, J=6.4 Hz, 4H), 3.21 (t, J=6.4 Hz, 4H). MS (ES⁺, m/z):(M+H)⁺: 413.4.

16.4-(trans-Isopropoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-16)

EX. 8-16 was prepared by similar procedures as in EX. 8-1 usingtrans-4-isopropoxycyclohexanamine.

¹H-NMR (CD₃OD/400 MHz): δ 8.35 (s, 1H), 7.98 (d, J=9.6 Hz, 1H), 7.84 (s,1H), 7.59 (d, J=9.6 Hz, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.20 (d, J=7.0 Hz,1H), 6.68 (d, J=9.6 Hz, 1H), 3.45 (m, 1H), 3.42 (m, 2H), 2.02 (m, 2H),1.33 (m, 2H), 1.14 (d, J=6.0 Hz, 6H). MS (ES⁺, m/z): (M+H)⁺: 435.5.

17.N-((4,4-difluorocyclohexyl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-17)

To a solution of DAST (7.7 mL) in 75 mL CCl₄ was added the compoundethyl 4-oxocyclohexanecarboxylate (5 g, 29 mmol) dropwise at 0° C., thenthe mixture was stirred for 16 h at r.t, water was added slowly and theorganic phase washed with water, dried over Na₂SO₄, distilled to affordthe compound ethyl 4,4-difluorocyclohexanecarboxylate (4.2 g, 71%) as acolorless oil.

To a solution of compound ethyl 4,4-difluorocyclohexanecarboxylate (4.2g, 21.8 mmol) in 40 mL EtOH was treated with 2N NaOH (1.34 g, 32.8 mmol)at 0° C., and the mixture was allowed to warm to r.t. and stirred for 18h. The mixture was diluted with water and the pH was adjusted to 3-4with 6N HCl. The mixture was extracted with toluene, dried andconcentrated to give the title compound4,4-difluorocyclohexanecarboxylic acid (3.4 g, 94%) as a white solid.

¹H-NMR (CDCl₃/400 MHz): δ 2.28-2.27 (m, 1H), 2.22-2.20 (m, 1H),2.08-2.02 (m, 3H), 1.99-1.94 (m, 3H), 1.19 (s, 1H).

To a solution of compound 4,4-difluorocyclohexanecarboxylic acid (3.5 g,21 mmol) in 80 mL THF, 4-methylmorpholine (2.51 g, 21 mmol) was added at−70° C., followed by isobutyl chloridocarbonate (2.85 g, 21 mmol). 30min later, 10 mL ammonium hydroxide was added. The resulting mixture wasallowed to warm up to 0° C. After removal of all solvents, the residuewas washed with water, PE to afford the compound4,4-difluorocyclohexanecarboxamide (1.82 g, 40%) as a white solid.

¹H-NMR (DMSO-d₆/400 MHz): δ 7.26 (s, 1H), 6.77 (s, 1H), 2.25-2.15 (m,1H), 2.05-1.95 (m, 2H), 1.80-1.75 (m, 4H), 1.57-1.53 (m, 2H).

To a solution of compound 4,4-difluorocyclohexanecarboxamide (1.8 g, 13mmol) in 100 mL THF was added LiBH₄ (1.1 g, 50 mmol) under N₂ stirredfor overnight. Then the reaction mixture was refluxed for 4 h. Aftercooled to r.t, it was poured into ice-water slowly. After filtration,the product was extracted with DCM from filtrate. Combined organiclayers washed with water, brine, dried over Na₂SO₄, filtered andcondensed to afford compound (4,4-difluorocyclohexyl)methanamine (0.8 g,crude) as a colorless oil.

To the solution of compound (4,4-difluorocyclohexyl)methanamine (100 mg,6 mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (190 mg,6 mmol) in 3 mL DMSO was added DIEA (0.21 mL, 12 mmol) and 10 mg CsF,the solvent was stirred for 5 h at 120° C., Then the mixture waspurified by HPLC to afford the compoundN-((4,4-difluorocyclohexyl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(21 mg, 10%) as a white solid.

¹H-NMR (CD3OD/400 MHz): δ 8.35 (s, 1H), 7.94-7.91 (t, J=13.2 Hz, 1H),7.93-7.82 (d, J=4 Hz, 1H), 7.62-7.60 (t, J=4.8 Hz, 1H), 7.49-7.46 (m,1H), 7.20-7.18 (t, J=2.8 Hz, 1H), 7.73-7.69 (d, 1H), 3.26-3.22 (t, J=6.4Hz, 2H), 2.05 (s, 1H), 2.02 (s, 2H), 1.93-1.65 (m, 4H), 1.38-1.22 (m,2H). MS (ES⁺, m/z): (M+H)⁺: 427.4.

18.N-((tetrahydro-2H-thiopyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-18)

To the solution of compound (tetrahydro-2H-thiopyran-4-yl)methanamine(150 mg, 1.14 mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (358 mg,1.14 mmol) in 3 mL DMSO was added DIEA (0.22 mL, 2.28 mmol) and 10 mgCsF, the solvent was stirred for 5 h at 120° C., Then the mixture waspurified by HPLC to afford the compoundN-((tetrahydro-2H-thiopyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(26 mg, 30%) as yellow oil.

¹H-NMR (CDCl₃/400 MHz): δ 8.29 (s, 1H), 8.23 (s, 1H), 7.98-7.95 (m, 2H),7.64-7.60 (t, J=8 Hz, 1H), 7.41-7.39 (d, J=8 Hz, 1H), 7.25-7.23 (d, J=10Hz, 1H), 3.24-3.22 (d, J=6.8 Hz, 2H), 2.68-2.55 (m, 4H), 2.14-2.10 (m,2H), 1.80-1.78 (t, J=3.2 Hz, 1H), 1.44-1.37 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 409.5.

19.N-((tetrahydro-2H-pyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-19)

Compound 6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine(0.2 g, 0.638 mmol), 4-aminomethyltetrahydropyran (0.145 g, 0.956 mmol),Sodium tertiarybutyloxide (0.172 g, 1.785 mmol), rac-BINAP (0.024 g,0.038 mmol) and Pd2(dba)3 (0.018 g, 0.019 mmol) were combined in a 20 mlvial. Toluene (5 mL) was added and nitrogen was bubbled through themixture for 5 minutes. The reaction mixture was then heated to 100° C.overnight. Silica gel was added to the reaction mixture and the solventstripped off. The product was isolated via column chromatography(hexanes:EtOAc) with the CompoundN-((tetrahydro-2H-pyran-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amineeluting at 100% EtOAc.

¹H-NMR (CDCl₃/400 MHz): δ 8.50 (s, 1H), 8.07 (d, J=7.6 Hz, 1H), 8.03 (s,1H), 7.77 (d, J=9.2 Hz, 1H), 7.58 (t, J=8.0 Hz, 1H), 7.29 (d, J=8.4 Hz,1H), 7.24 (bs, 1H), 6.76 (d, J=10.0 Hz, 1H), 3.87 (d, J=11.2 Hz, 1H),3.30 (m, 3H), 3.20 (m, 2H), 1.97 (bs, 1H), 1.69 (d, J=12.8 Hz, 2H), 1.26(m, 2H). MS (ES⁺, m/z): (M+H)⁺: 393.3

20.2-Hydroxy-2-methyl-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)propan-1-one(EX. 8-20)

CompoundN-(piperidin-4-ylmethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(200 mg, 0.47 mmol) was combined with 2-hydroxy-2-methylpropanoic acid(49 mg, 0.47 mmol), EDC (99 mg, 0.51 mmol) and HOAT (64 mg, 0.47 mmol).The mixture was then taken up in DMF (2 mL) and NMM (257 uL, 2.34 mmol)was added. The mixture was then stirred overnight at room temperature.The DMF solution was then poured into an excess of water (20 mL) causinga precipitate to form. The precipitate was collected by filtration andpurified via column chromatography (DCM/MeOH 0-15%) to give Compound2-hydroxy-2-methyl-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)propan-1-one.

¹H-NMR (CDCl₃/400 MHz): δ 8.46 (s, 1H), 8.14 (s, 1H) 8.08 (d, J=7.6 Hz,1H), 7.83 (d, J=9.2 Hz, 1H), 7.60 (t, J=8.0 Hz, 1H), 7.42 (bs, 1H), 7.32(d, J=8.0 Hz, 1H), 6.85 (d, J=9.2 Hz, 1H), 4.62 (m, 1H), 3.19 (s, 2H),2.99 (m, 1H), 2.76 (d, J=15.6 Hz, 2H), 2.66 (d, J=15.6 Hz, 2H), 1.99 (m,1H), 1.80 (d, J=13.2 Hz, 2H), 1.30 (s, 6H), 1.14 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 478.3

21.2-Morpholino-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)ethanone(EX. 8-21)

CompoundN-(piperidin-4-ylmethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(200 mg, 0.47 mmol) was combined with 2-morpholinoacetic acid (68 mg,0.47 mmol), EDC (99 mg, 0.51 mmol) and HOAT (64 mg, 0.47 mmol). Themixture was then taken up in DMF (2 mL) and NMM (257 uL, 2.34 mmol) wasadded. The mixture was then stirred overnight at room temperature. TheDMF solution was then poured into an excess of water (20 mL) causing aprecipitate to form. The precipitate was collected by filtration andpurified via column chromatography (DCM/MeOH 0-15%) The crude productwas then dissolved in methanolic HCl and precipitated with diethyl etherto give2-morpholino-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)ethanoneas the HCl salt.

¹H-NMR (CDCl₃/400 MHz): δ 10.26 (bs, 1H), 8.60 (s, 1H), 8.38 (m, 2H)8.09 (m, 2H), 7.69 (t, J=8.0 Hz, 1H), 7.48 (d, J=8.0 Hz, 1H), 7.39 (d,J=13.6 Hz, 1H), 4.62 (m, 4H), 3.90 (m, 5H), 3.68 (d, J=12.8 Hz, 1H),3.38 (m, 3H), 3.05 (t, J=12.0 Hz, 1H), 2.68 (t, J=12.0 Hz, 1H), 2.03 (m,1H), 1.86 (d, J=11.6 Hz, 2H), 1.26 (m, 1H), 1.09 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 519.3

22.1-Methyl-4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-4-ol(EX. 8-22)

CH₃NO₂ (6.6 mL, 122 mmol) and MeONa (221 mg, 4.1 mmol) was added to asolution of compound 1-methylpiperidin-4-one (10 mL, 81.3 mmol) in EtOH(10 mL). After 30 min more ethanol (15 mL) was added to facilitatestirring. The reaction mixture was stirred at room temperature for 2days and then filtered through celite. The isolated solid was washedwith ether to give the product 1-methyl-4-(nitromethyl)piperidin-4-ol(5.7 g, yield 40.4%). ¹H-NMR (DMSO-d₆/400 MHz): δ 5.01 (s, 1H), 4.47 (s,2H), 2.41-2.40 (m, 2H), 2.21-2.15 (m, 2H), 2.12 (s, 3H), 1.64-1.53 (m,4H).

A mixture of 1-methyl-4-(nitromethyl)piperidin-4-ol (0.6 g, 3.5 mmol)and Raney Ni (0.1 g) in methanol (25 ml) was stirred for 16 hrs at roomtemperature under ambient pressure of hydrogen gas. The mixture wasfiltered through celite and evaporated under reduced pressure. Theresidue was used without further purification4-(aminomethyl)-1-methylpiperidin-4-ol (0.4 g, 74%).

CsF (50 mg, 0.33 mmol) was added to the mixture of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (150 mg,0.48 mmol) and 4-(aminomethyl)-1-methylpiperidin-4-ol (150 mg, 1.04mmol) in DMSO (1.5 mL). The mixture was heated to 120° C. over night andpartitioned with EtOAc (10 mL) and water (10 mL). The organic layer waswashed with brine (8 mL×3), dried with Na₂SO₄ and concentrated underreduced pressure. The residue was purified by pre-HPLC to give theproduct1-methyl-4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-4-ol(22 mg, 10%)

¹H-NMR (CD₃OD/400 MHz): δ 8.28 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.84 (s,1H), 7.64 (d, J=5.6 Hz, 1H), 7.55 (t, J=8.0 Hz, 1H), 7.22 (d, J=8.0 Hz,1H), 6.84 (d, J=10.0 Hz, 1H), 3.47 (s, 2H), 2.58-2.60 (brs, 2H),2.48-2.47 (brs, 2H), 2.25 (s, 3H), 1.79-1.69 (m, 4H). MS (ES⁺, m/z):(M+H)⁺: 422.0.

23.4-(((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-2-one(EX. 8-23)

A solution of tert-butyl 2-oxopiperidine-1-carboxylate (5.4 g, 27 mmol)in THF (100 mL) was added LDA (16.2 mL, 32.4 mmol) at −78° C. Afterstirring for 0.5 h, phenyl selenisum chloride (7.94 g, 41.5 mmol) wasadded. The mixture was stirred at −78° C. for 4.5 hrs and then quenchedwith H₂O (30 mL), diluted with brine (200 mL). The aqueous layer wasextracted with CH₂Cl₂ (200 mL×2). The combined organic phase was dried,filtered and condensed. The residue was purified by flash chromatography(100% petroleum ether to petroleum ether/EtOAc=5:1) to give compoundtert-butyl 2-oxo-3-(phenylselanyl)piperidine-1-carboxylate (4.27 g, 45%)as an orange solid.

A solution of 30% H₂O₂ (2.2 mL, 36 mmol) was added to a stirringsolution of tert-butyl 2-oxo-3-(phenylselanyl)piperidine-1-carboxylate(4.27 g, 12 mmol) in THF (20 mL) at 0° C. The reaction mixture wasstirred 15 min at 0° C. and allowed to warm up to room temperature andkept for 30 min. The reaction mixture was then dissolved in CH₂Cl₂ (200mL) and washed with a saturated NaHCO₃. The organic phase was dried andcondensed to compound tert-butyl2-oxo-5,6-dihydropyridine-1(2H)-carboxylate (2.1 g, 88.6%).

A solution of tert-butyl 2-oxo-5,6-dihydropyridine-1(2H)-carboxylate(2.1 g, 11 mmol) in CH₃NO₂ (22.7 g, 372 mmol) under N₂ was added DBU(2.52 g, 16.5 mmol). The mixture was stirred at room temperatureovernight and then condensed and purified by flash chromatography togive compound tert-butyl 4-(nitromethyl)-2-oxopiperidine-1-carboxylate(0.6 g, 22%) as a white solid.

A solution of compound tert-butyl4-(nitromethyl)-2-oxopiperidine-1-carboxylate (0.3 g, 1.2 mmol), Ni (0.1g) and ammonia (2 mL) in MeOH (30 mL) was stirred under H₂ at roomtemperature for 7 hrs. The mixture was filtered and condensed tocompound tert-butyl 4-(aminomethyl)-2-oxopiperidine-1-carboxylate (0.13g, 49%) as a white solid.

A mixture of compound tert-butyl4-(aminomethyl)-2-oxopiperidine-1-carboxylate (37 mg, 0.16 mmol), DIEA(0.6 mL, 3.5 mmol),6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (50 mg)and CsF (17 mg, 0.11 mmol) in DMSO (2 mL) was stirred at 120° C.overnight. The mixture was diluted with H₂O (20 mL) and CH₂Cl₂ (20 mL).The organic phase was separated and condensed to give crude compoundtert-butyl2-oxo-4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidine-1-carboxylate(60 mg).

A solution of compound tert-butyl2-oxo-4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidine-1-carboxylate(114 mg, 0.23 mmol) in CH₂Cl₂ (10 mL) was added TFA (2 mL). The mixturewas stirred at room temperature for 30 min and condensed. The residuewas purified by prep-HPLC to give4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-2-one(6 mg, 6.6%) as white solid.

¹H-NMR (CD₃OD/400 MHz): δ 9.06 (d, J=10 Hz, 2H), 8.59 (s, 1H), 8.45 (d,J=10 Hz, 1H), 8.19 (s, 1H), 8.03-8.01 (m, 1H), 7.69 (t, J=8 Hz, 1H),7.51-7.48 (m, 1H), 4.36-4.32 (m, 1H), 3.79-3.74 (m, 1H), 3.06-3.01 (m,2H), 2.69-2.63 (m, 1H), 2.59-2.53 (m, 1H), 2.02-1.97 (m, 1H), 1.95-1.89(m, 1H). MS (ES⁺, m/z): 406.3.

24.4-(Aminomethyl)-1-(3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-2-one(EX. 8-24)

A solution of compound tert-butyl2-oxo-4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidine-1-carboxylate(114 mg, 0.23 mmol) in CH₂Cl₂ (10 mL) was added TFA (2 mL). The mixturewas stirred at room temperature for 30 min and condensed. The residuewas purified by prep-HPLC to give4-(aminomethyl)-1-(3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-2-oneas white solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.33 (s, 1H), 8.22 (s, 1H), 7.99 (m, 2H), 7.69(t, J=8.0 Hz, 1H), 7.44 (d, J=8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 3.52(m, 2H), 3.44 (m, 2H), 3.07 (m, 1H), 2.60 (m, 1H), 2.49 (m, 1H), 2.08(m, 1H), 1.86 (m, 1H). MS (ES⁺, m/z): 406.3.

25.(S)-1-(3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-3-amine(EX. 8-25)

EX. 8-25 was prepared by similar procedures as in EX. 8-1 using(S)-tert-butyl piperidin-3-ylcarbamate, and then deprotected with HCl indioxane.

¹H-NMR (CD₃OD/400 MHz): δ 8.22 (s, 1H), 7.96 (d, J=8.0 Hz, 1H), 7.89 (s,1H), 7.74 (d, J=8.0 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.20 (m, 2H), 3.98(m, 2H), 3.07 (m, 1H), 2.88 (m, 2H), 2.00 (m, 1H), 1.88 (m, 1H), 1.69(m, 1H), 1.43 (m, 1H). MS (ES⁺, m/z): (M+H)⁺: 378.5.

26.4-(trans-Hydroxycyclohexyl)amino)imidazo[1,2-b]pyridazin-3-yl)benzonitrile(EX. 8-26)

To a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine (2 g, 8.6mmol) and 3-(cyano)phenylboronic acid (1.26 g, 8.6 mmol) in dioxane/H₂O(100 mL, 4:1) was added K₂CO₃ (2.38 g, 17.2 mmol) and Pd(PPh₃)₄ (0.19 g,0.172 mmol), the mixture was stirred at 110° C. for 3 h. The solutionwas concentrated and partitioned in EtOAc/H₂O. The aqueous layer waswashed with EtOAc (50 mL) 3 times. The collected organic layers weredried over Na₂SO₄, concentrated and purified by column chromatography togive compound 3-(6-chloroimidazo[1,2-b]pyridazin-3-yl)benzonitrile (1.2g, 4.71 mmol, yield 55%) as a brown solid.

¹H-NMR (CDCl₃/400 MHz): δ 8.39 (s, 1H), 8.24 (m, 1H), 8.10 (s, 1H), 7.99(d, J=9.6 Hz, 1H), 7.65 (m, 2H), 7.15 (d, J=9.6 Hz, 1H). MS (ES⁺, m/z):(M+H)⁺: 264.3.

To a solution of 3-(6-chloroimidazo[1,2-b]pyridazin-3-yl)benzonitrile(300 mg, 1.18 mmol) and trans-4-aminocyclohexanol hydrogenchloride (678mg, 5.89 mmol) in NMP (2.0 mL) was added NaHCO₃ (496 mg, 5.89 mmol), themixture was stirred at 180° C. for 45 mins under microwave irridation.The mixture was purified by flash chromatograph to give 4-((3-(3-cyanophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanol (110 mg, 28%) asa pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.72 (s, 1H), 8.33 (m, 1H), 7.87 (s, 1H), 7.64(m, 3H), 6.70 (d, J=9.6 Hz, 1H), 3.61 (m, 2H), 2.23 (d, J=8.8 Hz, 2H),2.02 (d, J=8.8 Hz, 2H), 1.49 (m, 2H), 1.34 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 334.5.

27.2-(4-(((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)ethanol(EX. 8-27)

Bromoethanol (1.36 g, 11 mmol) and piperidine-4-carbonitrile (1.0 g, 9.1mmol) 5 mL of dimethylformamide. After this, 4.0 g of anhydrous sodiumcarbonate and 0.08 g of sodium iodide were added to the resultingsolution, and the mixture was stirred for 4 hours at 130° C. Then themixture was poured into ice water and extracted with ethyl acetate. Theoily extract obtained was purified by silica gel chromatography1-(2-hydroxyethyl)piperidine-4-carbonitrile (300 mg, 18%).

¹H-NMR (CDCl₃/400 MHz): δ 3.58 (t, J=4.6 Hz, 2H), 2.60-2.75 (m, 4H),2.50-2.60 (m, 2H), 2.31-2.46 (m, 2H), 1.70-2.00 (m, 4H).

To the solution of compound 1-(2-hydroxyethyl)piperidine-4-carbonitrile(300 mg, 2 mmol) in dry THF (5 mL) at 0° C., BH₃.SMe₂ (220 mg, 2.4 mmol)was added dropwise. The mixture was stirred at 0° C. for 2 hrs. Waterwas added then K₂CO₃. The mixture was filtered and concentrated to givecompound 2-(4-(aminomethyl)piperidin-1-yl)ethanol (150 mg, 47%).

¹H-NMR (CDCl₃/400 MHz): δ 3.61-3.70 (m, 1H), 3.59 (t, J=5.6 Hz, 2H),2.91 (d, J=11.6 Hz, 2H), 2.57 (d, J=6.4 Hz, 2H), 2.50 (t, J=5.6 Hz, 2H),1.97-2.10 (m, 2H), 1.63-1.70 (m, 2H), 1.15-1.35 (m, 2H).

To the solution of compound 2-(4-(aminomethyl)piperidin-1-yl)ethanol(150 mg, 0.95 mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (200 mg,0.64 mmol) in 3 mL DMSO was added DIEA (0.3 mL, 1.37 mmol) and 10 mgCsF, the solvent was stirred for 5 h at 120° C., Then the mixture waspurified by HPLC to afford the compound2-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)cyclohexyl)ethanol(34 mg, 8.2%) as a brown solid.

¹H-NMR (CDCl₃/400 MHz): δ 8.61 (s, 1H), 8.20-8.25 (m, 1H), 7.95-8.05 (m,2H), 7.66 (t, J=4.4 Hz, 1H), 7.21-7.28 (m, 1H), 3.80-3.90 (m, 2H),3.62-3.75 (m, 2H), 3.37 (d, J=6.4 Hz, 2H), 3.21 (t, J=5.2 Hz, 2H),2.91-3.09 (m, 2H), 2.10 (d, J=14.0 Hz, 2H), 1.51-1.69 (m, 2H). MS (ES⁺,m/z): (M+H)⁺: 435.5.

28.6-((Tetrahydro-2H-pyran-4-yl)methoxy)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine(EX. 8-28)

To the solution of compound (tetrahydro-2H-pyran-4-yl)methanol (100 mg,0.86 mmol) and compound6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (270 mg,0.86 mmol) in 10 mL dioxane was added t-BuOK (200 mg, 1.78 mmol). Themixture was stirred at 70° C. for 4 hrs. Then the mixture was purifiedby Pre-HPLC to afford the compound6-((tetrahydro-2H-pyran-4-yl)methoxy)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine(25 mg, 12%) as a white solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.18 (s, 1H), 7.89 (s, 1H), 7.82 (d, J=9.2 Hz,1H), 7.71-7.79 (m, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.10-7.19 (m, 1H), 6.69(d, J=9.6 Hz, 1H), 4.16 (d, J=6.8 Hz, 2H), 3.90-4.04 (m, 2H), 3.30-3.45(m, 2H), 2.01-2.15 (m, 1H), 1.63-1.75 (m, 2), 1.37-1.45 (m, 2H). MS(ES⁺, m/z): (M+H)⁺: 334.5.

29.4-((3-(3-(Trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexanol(EX. 8-29)

To a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine (2 g, 8.6mmol) and 3-(trifluoromethyl)phenylboronic acid (1.63 g, 8.6 mmol) indioxane/H₂O (100 mL, 4:1) was added K₂CO₃ (2.4 g, 17.2 mmol) andPd(PPh₃) 4 (0.497 g, 0.430 mmol), the mixture was stirred at 110° C. for3 h. The solution was concentrated, partitioned in EtOAc/H₂O. Theaqueous layer was washed with EtOAc (50 mL) for 3 times. The collectedorganic layers were dried over Na₂SO₄, concentrated and purified bycolumn chromatography to give compound6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (0.22 g,0.739 mmol, yield 9%) as a brown solid.

¹H-NMR (CDCl₃/400 MHz): δ 8.28 (s, 1H), 8.24 (m, 1H), 8.11 (s, 1H), 7.99(d, J=9.6 Hz, 1H), 7.64 (m, 2H), 7.13 (d, J=9.6 Hz, 1H).

To a solution of6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (220 mg,0.739 mmol) and trans-4-aminocyclohexanol hydrogenchloride (85 mg, 0.739mmol) in NMP (2.0 mL) was added NaHCO₃ (79 mg, 0.739 mmol), the mixturewas stirred at 180° C. for 45 mins under microwave irridation. Themixture was purified by flash chromatograph to give4-((3-(3-trifluoromethylphenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanol(40 mg, 0.106 mmol, 14%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.71 (s, 1H), 8.21 (m, 1H), 7.84 (s, 1H), 7.60(m, 3H), 6.67 (m, 1H), 3.71 (m, 1H), 3.59 (m, 1H), 2.18 (m, 2H), 1.96(m, 2H), 1.38 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 377.4.

EX. 8-29 free base and hydrochloride salt were prepared in 50 g scaleaccording the following procedures.

To a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine (150 g, 0.65mol) and 3-(trifluoromethyl)phenylboronic (143 g, 0.65 mol) indioxane/H₂O (2.5 L: 4:1) was added K₂CO₃ (180 g, 1.3 mol) and Pd(PPh₃)₄(10 g, 10 mmol). The mixture was stirred at 80° C. for 24 h. Thesolution was concentrated, partitioned with EA/H₂O. The aqueous layerwas extracted with EA (500 mL) for 3 times. The collected organic layerswas dried over Na₂SO₄, concentrated and purified by column chromatograph(PE:EA, 3:1 to 2:1) to give6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (135.15g, 70.3%) as yellow solid.

To a solution of6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (90 g,300 mmol) and trans-4-aminocyclohexanol (60 g, 522 mmol) in 300 mL ofDMSO was added DIEA (90 g, 900 mmol) and CsF (45 g, 30 mmol). Themixture was stirred at 180° C. for 4 hour. After cooled to the roomtemperature, the reaction mixture was poured into 3 L of water. Thesolid precipitated was collected, washed with water and recrystalizedwith MeOH to give the EX. 8-29 (Free Base) (68.3 g, 63.06%) as a whitesolid. The product precipated from MeOH generally contains 1 eq of MeOH.To remove the residue MeOH, the product was dissolved in EA, and thenre-evaporated.

To a solution of EX. 8-29 (Free Base) (54 g, 144 mmol) in EA (3000 mL)was added HCl/EA until no further solid formed. The solid was collectedand dried under vacuo to give EX. 8-29 (HCl Salt) (53 g, 98.13%) as awhite solid.

¹H NMR (MeOD/400 MHz): δ 8.64 (s, 1H), 8.37 (s, 1H), 8.27 (d, J=8.0 Hz,1H), 8.00 (d, J=9.6 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.79 (t, J=8.0 Hz,1H), 7.27 (d, J=9.6 Hz, 1H), 3.76-3.71 (m, 1H), 3.66-3.61 (m, 1H),2.22-2.20 (m, 2H), 2.03-2.01 (m, 2H), 1.45-1.41 (m, 4H). MS (ES⁺, m/z):(M+H)⁺: 377.2.

30.1-Methyl-4-((3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexanol(EX. 8-30)

EX. 8-30 was prepared by similar procedures as in EX. 8-1 usingtrans-4-amino-1-methyl cyclohexanol.

¹H-NMR (CD₃OD/400 MHz): δ 8.72 (s, 1H), 8.23 (d, J=7.6 Hz, 1H), 7.86 (s,1H), 7.62 (m, 3H), 6.74 (d, J=9.6 Hz, 1H), 3.84 (m, 1H), 2.08 (m, 2H),1.74 (m, 2H), 1.60 (m, 4H), 1.55 (s, 3H). MS (ES⁺, m/z): (M+H)⁺: 391.5.

31.4-((3-(3-(Trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-31)

EX. 8-31 was prepared by similar procedures as in EX. 8-1 using2-(trans-4-aminocyclohexyl)propan-2-ol.

¹H-NMR (CD₃OD/400 MHz): δ 8.82 (s, 1H), 8.19 (m, 1H), 7.88 (s, 1H), 7.62(m, 3H), 6.70 (d, J=9.6 Hz, 1H), 3.71 (m, 1H), 2.26 (m, 2H), 1.95 (m,2H), 1.36 (m, 1H), 1.27 (m, 4H), 1.21 (s, 6H). MS (ES⁺, m/z): (M+H)⁺:419.6.

Alternatively, EX. 8-31 was prepared in 50 g scale employing thefollowing procedures.

To a solution oftrans-4-((tert-butoxycarbonyl)amino)cyclohexanecarboxylic acid (823 g,3.38 mol) in EtOAc (4000 mL) was added EA/HCl (2500 mL). The mixture wasstirred at 0° C. overnight. The reaction mixture was filtered and driedin vacuo to give a product of hydrochloride salt oftrans-4-aminocyclohexanecarboxylic acid as white solid (604 g, 99.42%yield).

A mixture of hydrochloride salt of trans-4-aminocyclohexanecarboxylicacid (720 g), BnBr (1700 g, 2.5 eq) and K₂CO₃ in DMF (8000 mL) wasstirred at rt overnight. More BnBr (100 g) was added and the reactionmixture was heated to 50° C. and kept for 3 hrs. The reaction mixturewas then poured into water and extracted with EtOAc and combined organicphase washed with brine and concentrated in vacuo to give crudetrans-benzyl 4-(dibenzylamino)cyclohexanecarboxylate as white solid(1495 g, 93.9% yield).

To a solution of trans-benzyl 4-(dibenzylamino)cyclohexanecarboxylate(290 g×5, 3.6 mol) in 2 L of THF under N₂ at 0° C., MeMgBr (800 mL) wasadded. The mixture was stirred at room temperature overnight and thenquenched with 1.5 L of saturated NH₄Cl. The resulting mixture wasextracted with EtOAc. The product was extracted with 1 M HCl to theaqueous phase, which was then wash with EtOAc. The aqueous phase wasthen neutralized with NaOH, extracted with EtOAc, washed with brine,dried with Na₂SO₄ and concentrated in vacuo to give the2-(trans-4-(dibenzylamino)cyclohexyl)propan-2-ol as white solid (950 g,78.3% yield).

A mixture of 2-(trans-4-(dibenzylamino)cyclohexyl)propan-2-ol (120 g×8,356 mmol) and Pd(OH)₂ (15 g×8) in methanol (1000 mL) and MeOH/NH₃ (100mL) was stirred under H₂ (50 psi) at 50° C. for 72 hrs, then thecatalyst was removed and the filtrate was concentrated in vacuo and Thecrude product was chromatographed on silica gel (DCM/MeOH20:1-DCM/MeOH/NH₃ 5:4:1) to give the2-(trans-4-aminocyclohexyl)propan-2-ol as a pale yellow solid (210 g,47.5% yield).

6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine wasprepared according to procedure in EX. 8-29.

To a solution of6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (100 g,337 mmol) and 2-(trans-4-aminocyclohexyl)propan-2-ol (55 g, 350 mmol) in400 mL of DMSO was added DIEA (90 g, 900 mmol) and CsF (45 g, 30 mmol).The mixture was stirred at 180° C. for 4 hour. The solid was removed andthe filtrate was poured into a stirred solution of water (4 L) and EA (1L), The solid formed was collected and recrystallized from EA to giveEX. 8-31 (Free Base) as off white solid (70.58 g, 48.34%). From themother liquid and the filtrate, a second batch of product was obtainedafter column chromatography (EA).

¹H NMR (MeOD/400 MHz): δ 8.80 (s, 1H), 8.17 (d, J=6.8 Hz, 1H), 7.85 (s,1H), 7.62-7.58 (m, 3H), 6.68 (d, J=9.6 Hz, 1H), 3.72-3.65 (m, 1H),2.30-2.24 (m, 2H), 1.95-1.90 (m, 2H), 1.37-1.22 (m, 5H), 1.16 (s, 6H).MS (ES⁺, m/z): (M+H)⁺: 419.3. Melting Point: 216.7° C.-219.3° C.

To a production of EX. 8-31 (Free Base) (57 g, 136 mmol) in EA (10 L)was added HCl/EA until no further solid formed (about 100 mL of HCl/EA).The mixture was stirred at room temperature for half an hour and thesolid was collected, washed with EA and dried under vacuo to give EX.8-31 (HCl) (52.06 g, 91.33%) as off white solid.

¹H NMR (MeOD/400 MHz): δ 8.69 (s, 1H), 8.34 (s, 1H), 8.21 (d, J=7.6 Hz,1H), 7.96 (d, J=9.6 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.75 (dd, J=7.6 Hz,8.0 Hz, 1H), 7.23 (d, J=9.6 Hz, 1H), 3.73-3.66 (m, 1H), 2.24-2.20 (m,2H), 1.97 (m, 2H), 1.37-1.25 (m, 5H), 1.16 (s, 6H). MS (ES⁺, m/z):(M+H)⁺: 419.2. Melting Point: 200.4° C.-201.6° C.

EX. 8-32 to EX. 8-34 were prepared by similar procedures as in EX. 8-1.

32.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-32)

¹H-NMR (CD₃OD/400 MHz): δ 8.73 (s, 1H), 8.21 (d, J=8.0 Hz, 1H), 7.86 (s,1H), 7.60 (m, 3H), 6.69 (d, J=8.0 Hz, 1H), 3.73 (m, 1H), 3.34 (s, 3H),3.24 (m, 1H), 2.21 (m, 2H), 1.36 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 391.5.

33.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-33)

¹H-NMR (CD₃OD/400 MHz): δ 8.74 (s, 1H), 8.16 (d, J=6.8 Hz, 1H), 7.86 (s,1H), 7.62 (m, 3H), 6.71 (d, J=9.6 Hz, 1H), 3.98 (m, 3H), 3.53 (m, 2H),2.08 (m, 2H), 1.57 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 363.5.

34.4-(((3-(3-(Trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-34)

¹H-NMR (CD₃OD/400 MHz): δ 8.73 (s, 1H), 8.25 (d, J=8.0 Hz, 1H), 7.69 (s,1H), 7.64 (m, 3H), 6.76 (d, J=10.0 Hz, 1H), 3.98 (m, 2H), 3.06 (m, 4H),2.23 (m, 2H), 2.11 (m, 1H), 1.86 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 425.4.

35.4-((3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexanol(EX. 8-35)

To a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine (2 g, 8.6mmol) and 3-(chloro)phenylboronic acid (1.35 g, 8.6 mmol) in dioxane/H₂O(100 mL, 4:1) was added K₂CO₃ (2.4 g, 17.2 mmol) and Pd(PPh₃)₄ (0.19 g,0.172 mmol), the mixture was stirred at 110° C. for 3 h. The solutionwas concentrated, partitioned in EtOAc/H₂O. The aqueous layer was washedwith EtOAc (50 mL) for 3 times. The collected organic layers were driedover Na₂SO₄, concentrated and purified by column chromatography to givecompound 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (1.2 g,4.54 mmol, 53% yield) as a brown solid.

¹H-NMR (CDCl₃/400 MHz): δ 8.04 (d, J=13.2 Hz, 2H), 7.94 (m, 2H), 7.42(m, 2H), 7.10 (d, J=9.2 Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 264.3.

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine(300 mg, 1.14 mmol) and trans-4-aminocyclohexanol hydrogenchloride (327mg, 2.84 mmol) in NMP (2.0 mL) was added NaHCO₃ (286 mg, 3.41 mmol), themixture was stirred at 180° C. for 45 mins under microwave irridation.The mixture was purified by flash chromatograph to give4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanol(120 mg, 0.350 mmol, 31%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.47 (s, 1H), 7.90 (m, 1H), 7.80 (s, 1H), 7.58(d, J=10.0 Hz, 1H), 7.41 (m, 2H), 7.30 (m, 1H), 6.66 (m, 1H), 3.61 (m,2H), 2.20 (m, 2H), 2.00 (m, 2H), 1.50 (m, 2H), 1.45 (m, 2H). MS (ES⁺,m/z): (M+H)⁺: 343.5.

EX. 8-36 to EX. 8-40 were prepared by similar procedures as in EX. 8-35.

36.4-((3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-1-methyl-trans-cyclohexano(EX. 8-36)I

¹H-NMR (CD₃OD/400 MHz): δ 8.43 (s, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.79 (s,1H), 7.57 (d, J=8.0 Hz, 1H), 7.36 (m, 1H), 7.27 (d, J=8.0 Hz, 1H), 6.79(d, J=10.0 Hz, 1H), 3.80 (m, 1H), 2.14 (m, 2H), 1.73 (m, 4H), 1.52 (m,2H), 1.26 (s, 3H). MS (ES⁺, m/z): (M+H)⁺: 357.5.

37.4-((3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-37)

¹H-NMR (CD₃OD/400 MHz): δ 8.56 (s, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.82 (s,1H), 7.58 (d, J=9.6 Hz, 1H), 7.41 (m, 1H), 7.29 (d, J=6.8 Hz, 1H), 6.65(d, J=10.0 Hz, 1H), 3.68 (m, 1H), 2.30 (m, 2H), 1.94 (m, 4H), 1.30 (m,5H), 1.15 (s, 6H). MS (ES⁺, m/z): (M+H)⁺: 385.5.

38.3-(3-Chlorophenyl)-N-(4-trans-methoxycyclohexyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-38)

¹H-NMR (CD₃OD/400 MHz): δ 8.44 (s, 1H), 7.84 (d, J=9.6 Hz, 1H), 7.77 (s,1H), 7.55 (d, J=9.6 Hz, 1H), 7.35 (m, 1H), 7.26 (d, J=6.8 Hz, 1H), 6.63(d, J=10.0 Hz, 1H), 3.65 (m, 1H), 3.23 (m, 1H), 2.22 (m, 2H), 2.09 (m,2H), 1.32 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 357.5.

39.3-(3-Chlorophenyl)-N-(tetrahydro-2H-pyran-4-yl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-39)

¹H-NMR (CDCl₃/400 MHz): δ 8.35 (s, 1H), 7.81 (m, 2H), 7.74 (d, J=9.6 Hz,1H), 7.38 (m, 1H), 6.63 (d, J=9.6 Hz, 1H), 4.04 (m, 3H), 3.60 (m, 2H),2.17 (m, 2H), 1.60 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 329.5.

40.4-(((3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-40)

¹H-NMR (CD₃OD/400 MHz): δ 8.41 (s, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (s,1H), 7.63 (d, J=8.0 Hz, 1H), 7.40 (m, 1H), 7.30 (d, J=8.0 Hz, 1H), 6.68(d, J=8.0 Hz, 1H), 3.34 (m, 2H), 3.07 (m, 4H), 2.28 (m, 2H), 2.19 (m,1H), 1.85 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 391.4.

EX. 8-41 to EX. 8-42 were prepared by similar procedures as in EX. 8-4.

41.2-Methyl-4-((3-(3-(Trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)oxy)butan-2-ol(EX. 8-41)

¹H-NMR (CD₃OD/400 MHz): δ 8.41 (s, 1H), 7.94 (m, 2H), 7.82 (d, J=8.0 Hz,1H), 7.48 (t, J=8.0 Hz, 1H), 7.21 (d, J=9.6 Hz, 1H), 4.56 (t, J=6.4 Hz,1H), 2.05 (t, J=6.4 Hz, 1H), 1.32 (s, 6H). MS (ES⁺, m/z): (M+H)⁺: 382.5.

42.6-(2-Methoxyethoxy)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine(EX. 8-42)

¹H-NMR (CD₃OD/400 MHz): δ 8.11 (s, 1H), 7.98 (d, J=10.0 Hz, 1H), 7.94(s, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.49 (t, J=7.6 Hz, 1H), 7.22 (d, J=9.6Hz, 1H), 6.90 (d, J=9.6 Hz, 1H), 4.53 (t, J=4.8 Hz, 1H), 3.80 (t, J=4.8Hz, 1H), 3.45 (s, 3H). MS (ES⁺, m/z): (M+H)⁺: 354.4.

43.N-(4-Methoxybenzyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-43)

EX. 8-43 was prepared by similar procedures as in EX. 8-1.

¹H-NMR (CD₃OD/400 MHz): δ 8.21 (s, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.82 (s,1H), 7.64 (d, J=10.0 Hz, 1H), 7.48 (t, J=8.0 Hz, 1H), 7.32 (d, J=6.4 Hz,2H), 7.20 (d, J=8.4 Hz, 1H), 6.87 (m, 2H), 6.78 (d, J=10.0 Hz, 1H), 4.48(s, 2H), 3.75 (s, 3H). MS (ES⁺, m/z): (M+H)⁺: 415.5.

44.N-(2-morpholinoethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-44)

A solution of6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine (200 mg,0.638 mmol) and 2-morpholinoethanamine (83 mg, 0.638 mmol) in toluene (5mL, 0.638 mmol) was added sodium tetiarybutyloxide (110 mg, 1.148 mmol),rac-BINAP (23.81 mg, 0.038 mmol) and the mixture was heated at 100° C.for overnight. After 16 h, the resulting dark brown solution was cooleddown and was concentrated under reduced pressure. The solid was furtherpurified by using combiflash chromatography (12 g, DCM to 10% MeOH/DCM)gave 252 mg of yellow solidN-(2-morpholinoethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine.¹HNMR (400 MHz, CD₃OD) 8.28 (s, 1H), 8.00 (d, J=7.6 Hz, 1H), 7.82 (s,1H), 7.63 (d, J=9.6 Hz, 1H), 7.52 (m, 1H), 7.21 (d, J=8.0 Hz, 1H), 6.74(d, J=9.6 Hz, 1H), 3.67 (m, 4H), 3.53 (m, 2H), 2.68 (m, 2H), 2.52 (m,4H) ESI: 408.4 (M+H)⁺

The free base ofN-(2-morpholinoethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(258 mg, 0.658 mmol) was suspended into methanol (2 ml). ConcentratedHCl (137 uL, 12 M) was added and the solution was added drop wise into30 mL of Ether, filtration and dry to give 247 mg off white powder asHCl salt. ¹HNMR (400 MHz, CD₃OD) 8.31 (s, 1H), 8.26 (s, 2H), 7.97 (m,2H), 7.64 (m, 1H), 7.42 (d, J=8.21 Hz, 1H), 7.26 (m, 1H), 3.93 (m, 2H),3.35 (m, 4H), 2.02 (m, 1H), 1.71 (m, 2H), 1.34 (m, 2H).

45.N-(2-morpholinoethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-45)

The reaction mixture ofN-(piperidin-4-ylmethyl)-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-amine(100 mg 0.234 mmol), 2-(dimethylamino)acetyl chloride (43.5 mg, 0.234mmol), TEA (81 uL, 2.5 eq) in DMF (1 mL) was stirred overnight. Theresidue was poured into water (20 mL). The precipitate was collected byfiltration and purified by combiflash chromatography (12 g, DCM to 10%MeOH/DCM) gave 22 mg of solid2-(dimethylamino)-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)ethanone.¹HNMR (400 MHz, CD₃OD) 8.28 (m, 2H), 7.97 (m, 2H), 7.64 (t, J=8.02 Hz,1H), 7.41 (d, J=8.21 Hz, 1H), 7.22 (d, J=9.0 Hz, 1H), 4.53 (d, J=13.5Hz, 1H), 4.25 (m, 2H), 3.68 (d, J=13.5 Hz, 1H), 3.11 (m, 2H), 3.06 (s,3H), 2.90 (s, 3H), 2.71 (t, J=12.5 Hz, 2H), 2.08 (m, 1H), 1.90 (d,J=12.5 Hz, 2H). ESI: 477.5 (M+H)⁺

The free base2-(dimethylamino)-1-(4-(((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)piperidin-1-yl)ethanone(247 mg, 0.517 mmol) was suspended into methanol (2 mL). ConcentratedHCl (108 uL, 2.5 eq) was added. The solution was added dropwise into 30mL of Ether, filtration and dry to give 205 mg off white powder as HClsalt. ¹HNMR (400 MHz, CD₃OD) 8.29 (s, 1H), 8.25 (s, 1H), 7.96 (m, 2H),7.64 (t, J=8.02 Hz, 1H), 7.41 (d, J=8.41 Hz, 1H), 7.24 (d, J=9.8 Hz,1H), 2.02 (m, 1H), 1.87 (d, J=8.32 Hz, 2H), 1.39 (s, 6H).

46.4-((3-(3-(Trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexanol(EX. 8-46)

Synthesis of3-(dimethylamino)-2-(3-(trifluoromethoxy)phenyl)acrylonitrile

A mixture of 3-trifluoromethoxyphenylacetonitrile (2.5 grams, 12.43mmol), DIPEA (0.321 grams, 2.48 mmol), and dimethyl formamide dimethylacetal (20 mL) were heated at reflux for 4 h. On cooling, the reactionwas partitioned between EtOAc and saturated aqueous NH₄Cl solution. Theaqueous phase was extracted with ethyl acetate and the combined organicphase washed with brine and concentrated in vacuo. The crude product waspurified by chromatography (ethyl acetate/hexane, 0-10%) on silica gel(24 grams) to give pure product (2.3 grams, 8.98 mmol, 72% yield.).

Synthesis of 4-(3-(trifluoromethoxy)phenyl)-1H-pyrazol-5-amine

A mixture of acrylnitrile (2.0 g, 7.81 mmol), hydrazine hydrate (4.53grams, 39.0 mmol), and glacial acetaic acid (2.34 grams, 39.0 mmol) andethanol (20 mL) were heated at reflux for 16 h. On cooling, the reactionwas diluted with water, extracted with AcOEt and the combined organicphase washed with brine and concenrated in vacuo (1.90 grams, 7.84 mmol,100% yield.).

¹H-NMR (CDCl₃/400 MHz): δ 7.64 (m, 1H), 7.42 (m, 4H), 7.06 (d, J=7.6 Hz,1H). MS (ES⁺, m/z): (M+H)⁺: 244.3.

Synthesis of3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5(4H)-one

A mixture of pyrazole (0.8 grams, 3.29 mmol), 1,3-dimethyluracil (0.51grams, 3.62 mmol), and dry EtOH 10 mL were treated dropwise with sodiumethoxide (0.29 grams, 4.28 mmol) and on completion of addition thereaction was heated at reflux for 16 h. On cooling the reaction wasconcentrated in vacuo and the residue added to ice, neutralised withacetic acid and the resulting precipitate filtered, washed with waterand dried to give the product (0.4 grams, 1.36 mmol, 41% yield.). Nochromatography was needed for this step.

¹H-NMR (CDCl₃/400 MHz): δ 8.42 (d, J=8.0 Hz, 1H), 8.09 (s, 1H), 7.62 (d,J=7.6 Hz, 1H), 7.52 (m, 2H), 7.20 (d, J=8.8 Hz, 1H), 6.16 (d, J=7.6 Hz,1H).

Synthesis of5-chloro-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidine

A mixture of pyrazolepyrimidinone (0.5 grams, 1.69 mmol) was treatedwith POCl₃ 10 mL and the mixture was heated at relux for overnight. Oncooling, after remove the solvent under rotovapor, the reaction waspoured onto ice, cautiously made basic with saturated aqueous NaHCO₃solution and extracted with EtOAc. The combined organic phases werewashed with brine and concentrated in vacuo to give the product (0.3grams, 0.96 mmol, 56% yield.).

¹H-NMR (CDCl₃/400 MHz): δ 8.56 (d, J=7.6 Hz, 1H), 8.39 (s, 1H), 7.92 (d,J=7.6 Hz, 1H), 7.85 (m, 2H), 7.45 (t, J=8.0 Hz, 1H), 7.12 (d, J=8.4 Hz,1H), 6.85 (d, J=7.2 Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 314.3.

Synthesis of4-((3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexanol

A solution of 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine (0.03 grams,0.096 mmol) and trans-1,4-diamocyclohexane (0.017 grams, 0.143 mmol) iniso-propanol (5 mL) was added DIPEA (0.025 grams, 0.191 mmol) and themixture was heated at 150° C. for 12 h under microwave irradiation. Theresulting dark brown solution was cooled down and was concentrated underreduced pressure. The solid was further purified by using combiflashchromatography (12 g column), eluent: 0-6% CH₃OH/DCM and obtainedproduct (0.020 grams, 0.051 mmol, 53% yield.).

¹H-NMR (CD₃OD/400 MHz): δ 8.22 (s, 1H), 8.18 (m, 2H), 7.80 (d, J=8.0 Hz,1H), 7.34 (t, J=8.4 Hz, 1H), 6.96 (m, 1H), 6.17 (d, J=7.6 Hz, 1H), 3.90(m, 1H), 3.58 (m, 1H), 2.18 (m, 2H), 2.00 (m, 2H), 1.38 (m, 4H). MS(ES⁺, m/z): (M+H)⁺: 393.5.

EX. 8-47 to EX. 8-53 were prepared using similar procedures as in EX.8-46.

47.4-((3-(3-(Trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-47)

¹H-NMR (CD₃OD/400 MHz): δ 8.38 (s, 1H), 8.22 (m, 2H), 7.78 (d, J=8.0 Hz,1H), 7.38 (t, J=8.0 Hz, 1H), 6.97 (d, J=7.6 Hz, 1H), 6.22 (d, J=7.6 Hz,1H), 3.90 (m, 1H), 2.25 (m, 2H), 1.97 (m, 2H), 2.00 (m, 2H), 1.37 (m,1H), 1.30 (m, 4H), 1.16 (s, 6H). MS (ES⁺, m/z): (M+H)⁺: 435.5.

48.1-Methyl-4-((3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexanol(EX. 8-48)

¹H-NMR (CD₃OD/400 MHz): δ 8.25 (m, 3H), 7.95 (d, J=8.0 Hz, 1H), 7.40 (t,J=8.0 Hz, 1H), 6.99 (d, J=7.6 Hz, 1H), 6.29 (d, J=7.6 Hz, 1H), 4.10 (m,1H), 2.11 (m, 2H), 1.74 (m, 2H), 1.62 (m, 4H), 1.28 (s, 3H). MS (ES⁺,m/z): (M+H)⁺: 407.5.

49.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-49)

¹H-NMR (CD₃OD/400 MHz): δ 8.30 (m, 1H), 8.22 (m, 2H), 7.82 (d, J=8.0 Hz,1H), 7.38 (t, J=8.0 Hz, 1H), 6.38 (m, 1H), 6.22 (d, J=7.6 Hz, 1H), 3.95(m, 1H), 3.26 (m, 1H), 2.22 (m, 2H), 2.14 (m, 2H), 1.38 (m, 4H). MS(ES⁺, m/z): (M+H)⁺: 407.5.

50.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-50)

¹H-NMR (CDCl₃/400 MHz): δ 8.32 (s, 1H), 8.29 (m, 2H), 7.83 (d, J=8.0 Hz,1H), 7.41 (t, J=8.0 Hz, 1H), 7.02 (d, J=8.0 Hz, 1H), 6.29 (d, J=8.0 Hz,1H), 4.24 (m, 1H), 4.02 (m, 2H), 3.60 (t, J=12.0 Hz, 2H), 2.12 (m, 2H),1.62 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 379.4.

51.4-(((3-(3-(Trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-51)

¹H-NMR (CD₃OD/400 MHz): δ 8.24 (m, 2H), 8.21 (s, 1H), 7.70 (d, J=8.0 Hz,1H), 7.38 (d, J=8.0 Hz, 1H), 7.23 (m, 1H), 6.07 (d, J=7.2 Hz, 1H), 3.51(t, J=6.4 Hz, 2H), 3.06 (m, 2H), 3.00 (m, 2H), 2.22 (m, 3H), 1.96 (m,2H). MS (ES⁺, m/z): (M+H)⁺: 441.4.

52.N-(3-(3-(Trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)cyclohexane-1,4-trans-diamine(EX. 8-52)

¹H-NMR (CD₃OD/400 MHz): δ 8.24 (m, 3H), 7.87 (d, J=7.6 Hz, 1H), 7.39 (t,J=8.0 Hz, 1H), 6.99 (d, J=8.0 Hz, 1H), 6.24 (d, J=7.6 Hz, 1H), 3.95 (m,1H), 2.86 (m, 1H), 2.24 (m, 2H), 1.8 (m, 2H), 1.37 (t, J=9.2 Hz, 4H). MS(ES⁺, m/z): (M+H)⁺: 392.4.

53.(S)-1-(3-(3-(Trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-amine(EX. 8-53)

¹H-NMR (CD₃OD/400 MHz): δ 8.32 (d, J=8.0 Hz, 1H), 8.24 (m, 1H), 8.07 (m,1H), 7.85 (d, J=5.6 Hz, 1H), 7.38 (t, J=8.0 Hz, 1H), 6.95 (d, J=7.6 Hz,1H), 6.61 (d, J=7.6 Hz, 1H), 4.22 (m, 2H), 3.17 (m, 2H), 2.91 (m, 1H),2.10 (m, 1H), 1.80 (m, 1H), 1.60 (m, 1H), 1.50 (m, 1H). MS (ES⁺, m/z):(M+H)⁺: 378.5.

54.4-((3-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexanol(EX. 8-54)

A mixture of 2-(3-(trifluoromethyl)phenyl)acetonitrile (4.55 g, 24.58mmol), 1,1-dimethoxy-N,N-dimethylmethanamine (13.10 ml, 98 mmol), andN¹,N¹,N²,N²-tetramethylethane-1,2-diamine (0.737 ml, 4.92 mmol) wereheated to reflux for 5 h. After cooling to RT, the mixture waspartitioned between saturated aqueous NH₄Cl and EtOAc and extractedthree times with EtOAc. The combined organics were washed with brine,dried over Na₂SO₄, and concentrated in vacuo. After absorbing on celite,the compound was purified by Isco (40 g silica, 10% to 70%EtOAc/hexanes) to give pure3-(dimethylamino)-2-(3-(trifluoromethyl)phenyl)acrylonitrile (2.93 g,50% yield).

¹H NMR (400 MHz, CDCl₃): 7.48 (m, 2H), 7.41-7.34 (m, 2H), 6.94 (s, 1H),3.25 (s, 6H).

To a mixture of3-(dimethylamino)-2-(3-(trifluoromethyl)phenyl)acrylonitrile (2.93 g,12.20 mmol) in ethanol (35 ml) was added hydrazine hydrate (3.80 ml, 122mmol) and acetic acid (6.98 ml, 122 mmol). Upon to reflux; a solutionformed. After 5 hours, 0.25 mL of hydrazine hydrate was added and thereaction refluxed an additional 15 hours. The volatiles were removed invacuo, and the residue partitioned between EtOAc and water. Afterextracting three times with EtOAc, the combined organics were washedtwice with saturated aqueous NaHCO₃, dried over Na₂SO₄, and concentratedin vacuo to cleanly give4-(3-(trifluoromethyl)phenyl)-1H-pyrazol-5-amine (2.7 g, 97% yield) as alight yellow oil.

¹H NMR (400 MHz, CDCl₃): 7.69 (m, 1H), 7.63 (m, 1H), 7.58 (s, 1H), 7.50(m, 2H), 5.78 (br s, 3H).

To a mixture of 4-(3-(trifluoromethyl)phenyl)-1H-pyrazol-5-amine (2.7 g,11.88 mmol) and 1,3-dimethylpyrimidine-2,4(1H,3H)-dione (1.999 g, 14.26mmol) in ethanol (35 ml) was added sodium ethoxide (1.132 g, 16.64mmol). After heating to reflux 16 hours under Agonr, the reaction wascooled to RT and the volatiles removed in vacuo. The mixture was dilutedwith 30 mL H₂O and acidified to pH=4 with AcOH. The resulting solid wascollected by vacuum filtration. The solid was taken up in a 4:1DCM/2-propanol solution, washed with water, dried over Na₂SO₄, androtovaced to give pure3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5(4H)-one (2.07 g,7.41 mmol, 62.4% yield).

¹H NMR (400 MHz, DMSO-d6): 12.38 (br s, 1H), 8.62 (d, 1H), 8.38 (s, 1H),8.05 (m, 2H), 7.60 (m, 2H), 6.20 (m, 1H).

A mixture of3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5(4H)-one (2.07 g,7.41 mmol) and POCl₃ (6.91 ml, 74.1 mmol) was heated to 107° C. Theheating was continued for 16 hours. The reaction mixture was cooled toRT and the POCl₃ removed in vacuo to give a waxy solid. The solid wastriturated multiple times with Et₂O and the Et₂O layers were combinedand rotovaced. The resulting residue was taken up in DCM and washed withsaturated aqueous NaHCO₃, dried over Na₂SO₄ and concentrated in vacuo togive 5-chloro-3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine(1.47 g, 67% yield) as a brown solid.

¹H NMR (400 MHz, CDCl₃): 8.60 (d, 1H), 8.47 (s, 1H), 8.24 (d, 1H), 8.19(s, 1H), 7.55 (m, 2H), 6.87 (d, 1H).

A mixture of5-chloro-3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidine (100 mg,0.336 mmol), 4-aminocyclohexanol (58.0 mg, 0.504 mmol), and DIEA (0.117ml, 0.672 mmol) in 2-propanol (3 ml) was irradiated to 135° C. for 14 hin a Biotage microwave. After cooling, the mixture was diluted withsaturated aqueous NaHCO₃, and extracted three times with EtOAc. Thecombined extracts were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. After absorbing on celite, the compound waspurified by Isco (12 g silica, 1/5/4 MeOH/EtOAc/hex) to givetrans-4-((3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexanol(101 mg, 80% yield) as a white solid.

¹H NMR (400 MHz, CD₃OD): 8.72 (s, 1H), 8.29 (s, 1H), 8.50 (d, 1H), 8.09(d, 1H), 7.50 (t, 1H), 7.39 (d, 1H), 6.25 (d, 1H), 3.98 (m, 1H), 3.62(m, 1H), 2.23 (m, 2H), 2.03 (m, 2H), 1.55-1.32 (m, 4H). MS (ES⁺, m/z):(M+H)⁺: 377.5.

EX. 8-55 to EX. 8-60 were prepared using similar procedures as in EX.8-54.

55.1-Methyl-4-((3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexanol(EX. 8-55)

¹H NMR (400 MHz, CD₃OD): 8.69 (s, 1H), 8.29 (s, 1H), 8.25 (d, 1H), 8.11(d, 1H), 7.30 (t, 1H), 7.39 (d, 1H), 6.31 (d, 1H), 4.09 (m, 1H), 2.15(m, 2H), 1.79-1.52 (m, 6H), 1.30 (s, 3H). MS (ES⁺, m/z): (M+H)⁺: 391.5.

56.4-((3-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-56)

¹H NMR (400 MHz, CD₃OD): 8.78 (s, 1H), 8.18 (s, 1H), 8.24 (d, 1H), 8.05(d, 1H), 7.49 (t, 1H), 7.38 (d, 1H), 6.25 (d, 1H), 3.95 (m, 1H), 2.28(m, 2H), 1.95 (m, 2H), 1.32 (m, 5H), 1.20 (s, 6H). MS (ES⁺, m/z):(M+H)⁺: 419.5.

57.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-57)

¹H NMR (400 MHz, DMSO-d6): 8.76 (s, 1H), 8.49 (d, 1H), 8.45 (s, 1H),8.13 (d, 1H), 7.66 (d, 1H), 7.56 (t, 1H), 7.42 (d, 1H), 6.30 (d, 1H),3.75 (m, 1H), 3.27 (s, 3H), 3.19 (m. 1H), 2.10 (m, 4H), 1.30 (m, 4H). MS(ES⁺, m/z): (M+H)⁺: 391.5.

58.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-58)

¹H NMR (400 MHz, DMSO-d6): 8.73 (s, 1H), 8.52 (d, 1H), 8.47 (s, 1H),8.15 (d, 1H), 7.77 (d, 1H), 7.55 (t, 1H), 7.43 (d, 1H), 6.33 (d, 1H),4.08 (m, 1H), 3.93 (m, 2H), 3.43 (m, 2H), 2.05 (m, 2H), 1.53 (m, 2H). MS(ES⁺, m/z): (M+H)⁺: 363.5.

59.4-(((3-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-59)

¹H NMR (400 MHz, CD₃OD): 8.66 (s, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 8.12(d, 1H), 7.52 (t, 1H), 7.40 (d, 1H), 6.31 (d, 1H), 3.50 (d, 2H), 3.18(m, 4H), 2.21 (m, 3H), 1.88 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 425.5.

60.4-(3-(3-(Trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)thiomorpholine1,1-dioxide (EX. 8-60)

¹H NMR (400 MHz, DMSO-d6): 8.82 (d, 1H), 8.61 (s, 1H), 8.48 (m, 1H),8.28 (d, 1H), 7.61 (t, 1H), 7.48 (d, 1H), 6.98 (d, 1H), 4.22 (m, 4H),3.28 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 397.4.

61.4-((3-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexanol(EX. 8-61)

Synthesis of 3-(dimethylamino)-2-(3-chlorophenyl)acrylonitrile

A mixture of 3-chlorophenylacetonitrile (5 grams, 33.0 mmol),N,N,N′,N′-tetramethyl-ethane-1,2-diamine (0.767 grams, 6.60 mmol), anddimethyl formamide dimethyl acetal (20 mL) were heated at reflux for 4h. On cooling, the reaction was partitioned between EtOAc and saturatedaqueous NH₄Cl solution. The aqueous phase was extracted with ethylacetate and the combined organic phase washed with brine andconcentrated in vacuo. The crude product was purified by chromatography(ethyl acetate/hexane, 0-10%) on silica gel (24 grams) to give pureproduct (6.8 grams, 33.0 mmol, 100% yield.).

¹H-NMR (CDCl₃/400 MHz): δ 7.31 (m, 2H), 7.22 (m, 2H), 7.08 (m, 1H), 3.31(s, 3H), 3.30 (s, 3H).

Synthesis of 4-(3-chlorophenyl)-1H-pyrazol-5-amine

A mixture of acrylnitrile (5.0 g, 24.19 mmol), hydrazine hydrate (7.75grams, 242 mmol), and glacial acetaic acid (14.53 grams, 242 mmol) andethanol (20 mL) were heated at reflux for 16 h. On cooling, the reactionwas diluted with water, extracted with AcOEt and the combined organicphase washed with brine and concentrated in vacuo (4.0 grams, 20.66mmol, 85% yield.).

¹H-NMR (CDCl₃/400 MHz): δ 7.63 (m, 1H), 7.52 (m, 1H), 7.42 (d, J=8.0 Hz,1H), 7.33 (t, J=8.0 Hz, 1H), 7.17 (d, J=8.0 Hz, 1H). MS (ES⁺, m/z):(M+H)⁺: 194.3.

Synthesis of 3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5(4H)-one

A mixture of pyrazole (4.0 grams, 20.66 mmol), 1,3-dimethyluracil (3.47grams, 24.79 mmol), and dry EtOH 10 mL were treated dropwise with sodiumethoxide (1.97 grams, 28.9 mmol) and on completion of addition thereaction was heated at reflux for 16 h. On cooling the reaction wasconcentrated in vacuo and the residue added to ice, neutralised withacetic acid and the resulting precipitate filtered, washed with waterand dried to give the product (4.0 grams, 16.28 mmol, 79% yield.). Nochromatography was needed for this step.

¹H-NMR (CDCl₃/400 MHz): δ 8.38 (d, J=8.4 Hz, 1H), 8.04 (s, 1H), 7.65 (s,1H), 7.65 (s, 1H), 7.51 (m, 1H), 7.40 (t, J=8.0 Hz, 1H), 7.27 (d, J=8.0Hz, 1H), 6.12 (d, J=7.6 Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 246.3.

Synthesis of 5-chloro-3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidine

A mixture of pyrazolepyrimidinone (4.0 grams, 16.28 mmol) was treatedwith POCl₃ 10 mL and the mixture was heated at relux for overnight. Oncooling, after remove the solvent under rotovapor, the reaction waspoured onto ice, cautiously made basic with saturated aqueous NaHCO₃solution and extracted with EtOAc. The combined organic phases werewashed with brine and concentrated in vacuo to give the product (0.65grams, 2.46 mmol, 15% yield.).

¹H-NMR (CDCl₃/400 MHz): δ 8.89 (d, J=7.4 Hz, 1H), 8.61 (s, 1H), 8.10 (m,1H), 7.96 (d, J=7.6 Hz, 1H), 7.40 (t, J=8.0 Hz, 1H), 7.26 (d, J=8.0 Hz,1H), 7.06 (d, J=7.2 Hz, 1H). MS (ES⁺, m/z): (M)⁺: 264.3.

Synthesis of4-((3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexanol

A solution of 3-chloro-5-chloropyrazolo[1,5-a]pyrimidine (0.150 grams,0.568 mmol) and trans-1,4-diamocyclohexane (0.131 grams, 1.14 mmol) iniso-propanol (5 mL) was added DIPEA (0.177 grams, 2.272 mmol) and themixture was heated at 150° C. for 12 h under microwave irradiation. Theresulting dark brown solution was cooled down and was concentrated underreduced pressure. The solid was further purified by using combiflashchromatography (12 g column), eluent: 0-6% CH3OH/DCM and obtainedproduct (0.100 grams, 0.292 mmol, 52% yield.).

¹H-NMR (CD₃OD/400 MHz): δ 8.41 (s, 1H), 8.24 (d, J=8.0 Hz, 2H), 7.80 (d,J=8.0 Hz, 1H), 7.30 (t, J=8.4 Hz, 1H), 7.10 (m, 1H), 6.25 (d, J=7.6 Hz,1H), 3.95 (m, 1H), 3.63 (m, 1H), 2.26 (m, 2H), 2.04 (m, 2H), 1.55 (m,2H), 1.39 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 343.5.

EX. 8-62 to EX. 8-67 and EX. 8-69 were prepared using similar proceduresas in EX. 8-61.

62.4-((3-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-1-methyl-trans-cyclohexanol(EX. 8-62)

¹H-NMR (CD₃OD/400 MHz): δ 8.36 (s, 1H), 8.23 (m, 2H), 7.84 (d, J=8.0 Hz,1H), 7.30 (t, J=8.0 Hz, 1H), 7.11 (d, J=7.6 Hz, 1H), 6.29 (d, J=7.6 Hz,1H), 4.02 (m, 1H), 2.20 (m, 2H), 1.75 (m, 4H), 1.50 (m, 2H), 1.29 (s,6H). MS (ES⁺, m/z): (M+H)⁺: 357.5.

63.4-((3-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-63)

¹H-NMR (CD₃OD/400 MHz): δ 8.35 (s, 1H), 8.12 (m, 2H), 7.72 (d, J=8.0 Hz,1H), 7.27 (t, J=8.0 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 6.20 (d, J=7.6 Hz,1H), 3.95 (m, 1H), 2.30 (m, 2H), 1.94 (m, 2H), 1.32 (m, 4H), 1.50 (s,6H). MS (ES⁺, m/z): (M+H)⁺: 385.5.

64.3-((3-Chlorophenyl)-N-4-methoxy-trans-cyclohexyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-64)

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 8.23 (m, 2H), 7.80 (d, J=8.0 Hz,1H), 7.29 (t, J=8.0 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 6.22 (d, J=7.6 Hz,1H), 3.95 (m, 1H), 3.30 (m, 1H), 2.25 (m, 2H), 2.15 (m, 2H), 1.45 (m,4H). MS (ES⁺, m/z): (M+H)⁺: 357.5.

65.3-(3-Chlorophenyl)-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-65)

¹H-NMR (CDCl₃/400 MHz): δ 8.32 (s, 1H), 8.15 (m, 2H), 7.72 (d, J=8.0 Hz,1H), 7.26 (t, J=8.0 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.23 (d, J=8.0 Hz,1H), 4.20 (m, 1H), 4.00 (m, 2H), 3.62 (t, J=12.0 Hz, 2H), 2.17 (m, 2H),1.62 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 329.5.

66.4-(((3-(3-Chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-66)

¹H-NMR (CD₃OD/400 MHz): δ 8.35 (s, 1H), 8.28 (d, J=8.0 Hz, 1H), 8.24 (s,1H), 7.81 (d, J=8.0 Hz, 1H), 7.31 (t, J=7.2 Hz, 1H), 7.11 (d, J=7.2 Hz,1H), 6.29 (d, J=7.2 Hz, 1H), 3.46 (m, 2H), 3.10 (m, 4H), 2.27 (m, 3H),1.89 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 391.4.

67.N-((1-Methylpiperidin-4-yl)methyl)-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-amine(EX. 8-67)

¹H-NMR (CD₃OD/400 MHz): δ 8.26 (m, 3H), 7.86 (d, J=8.0 Hz, 1H), 7.37 (m,1H), 6.99 (d, J=8.0 Hz, 1H), 6.28 (d, J=7.2 Hz, 1H), 3.41 (d, J=6.0 Hz,2H), 2.80 (d, J=12.0 Hz, 1H), 2.30 (s, 3H), 2.11 (t, J=12.0 Hz, 1H),1.89 (m, 3H), 1.44 (m, 2H), 1.29 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 391.4.

68.N1-(3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-trans-diamine(EX. 8-68)

EX. 8-68 was prepared using similar procedures as in EX. 8-8.

¹H-NMR (CD₃OD/400 MHz): δ 8.42 (s, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.78 (s,1H), 7.59 (d, J=8.0 Hz, 1H), 7.37 (t, J=8.0 Hz, 1H), 7.27 (m, 1H), 6.66(d, J=7.6 Hz, 1H), 3.65 (m, 1H), 3.01 (m, 1H), 2.33 (m, 2H), 2.08 (m,2H), 1.56 (m, 2H), 1.38 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 325.5.

69.N-(3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)cyclohexane-1,4-trans-diamine(EX. 8-69)

¹H-NMR (CD₃OD/400 MHz): δ 8.37 (s, 1H), 8.23 (m, 2H), 7.80 (d, J=8.0 Hz,1H), 7.28 (t, J=7.6 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 6.23 (d, J=7.2 Hz,1H), 3.90 (m, 1H), 2.75 (m, 1H), 2.27 (m, 2H), 2.05 (m, 2H), 1.36 (m,4H). MS (ES⁺, m/z): (M+H)⁺: 325.5.

70.N¹-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,3-diamine(EX. 8-70)

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine(100 mg, 0.38 mmol) and cyclohexane-1,3-diamine (43 mg, 0.38 mmol) inNMP (1.0 mL) was added NaHCO₃ (32 mg, 0.38 mmol), the mixture wasstirred at 180° C. for 60 mins under microwave irridation. The mixturewas purified by flash chromatograph to giveN¹-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,3-diamine(30 mg, 0.088 mmol, 23%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.44 (s, 1H), 7.84 (d, 1H), 7.78 (m, 1H), 7.58(d, 1H), 7.28 (m, 1H), 7.25 (m, 1H), 6.65 (d, 1H), 3.75 (m, 1H), 2.94(m, 1H), 2.41 (m, 1H), 2.20 (m, 1H), 1.95 (m, 2H), 1.51 (m, 1H), 1.16(m, 3H). MS (ES⁺, m/z): (M+H)⁺: 342.5.

71.(S)-1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-3-amine(EX. 8-71)

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine(200 mg, 0.78 mmol) and (S)-Piperidin-3-amine (290 mg, 2.12 mmol) in NMP(1.0 mL) was added NaHCO₃ (118 mg, 1.52 mmol), the mixture was stirredat 180° C. for 60 mins under microwave irridation. The mixture waspurified by flash chromatograph to give(S)-1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)piperidin-3-amine(32 mg, 0.098 mmol, 13%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.19 (s, 1H), 7.87 (d, J=7.6 Hz, 1H), 7.79 (s,1H), 7.66 (d, J=10.0 Hz, 1H), 7.34 (t, J=8.0 Hz, 1H), 7.23 (d, J=8.0 Hz,1H), 7.07 (d, J=10.0 Hz, 1H), 3.97 (m, 1H), 3.95 (m, 1H), 2.99 (m, 1H),2.83 (m, 2H), 1.98 (m, 1H), 1.79 (m, 1H), 1.60 (m, 1H), 1.36 (m, 1H). MS(ES⁺, m/z): (M+H)⁺: 328.6.

72.4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(EX. 8-72)

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine(200 mg, 0.757 mmol) and 4-aminocyclohexanone (290 mg, 2.56 mmol) in NMP(1.0 mL) was added NaHCO₃ (118 mg, 1.52 mmol), the mixture was stirredat 180° C. for 60 mins under microwave irridation. The mixture waspurified by flash chromatograph to give4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(40 mg, 0.117 mmol, 16%) as a pale yellow solid.

¹H-NMR (CD₃Cl/400 MHz): δ 8.38 (s, 1H), 7.83 (s, 1H), 7.76 (d, J=7.6 Hz,2H), 7.37 (t, J=8.0 Hz, 1H), 7.29 (m, 1H), 6.64 (d, J=9.6 Hz, 1H), 4.90(m, 1H), 4.25 (m, 1H), 2.53 (m, 6H), 1.80 (m, 2H). MS (ES⁺, m/z):(M+H)⁺: 341.4.

73.4-trans-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(EX. 8-73)

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (80mg, 0.303 mmol) and trans-4-aminocyclohexanecarbonitrile (38 mg, 0.303mmol) in NMP (1.0 mL) was added NaHCO₃ (102 mg, 1.212 mmol), the mixturewas stirred at 180° C. for 60 mins under microwave irridation. Themixture was purified by flash chromatograph to give4-trans-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(20 mg, 0.057 mmol, 23%) as white solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 7.83 (d, 1H), 7.78 (s, 1H), 7.56(d, 1H), 7.38 (m, 1H), 7.29 (m, 1H), 6.63 (d, 1H), 3.67 (m, 1H), 2.65(m, 1H), 2.30 (m, 2H), 2.20 (m, 2H), 1.75 (m, 2H), 1.32 (m, 2H). MS(ES⁺, m/z): (M+H)⁺: 352.5.

75.2-(trans-4-((3-(3-fluorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)propan-2-ol(EX. 8-75)

To a solution of 3-bromo-6-chloroimidazo[1,2-b]pyridazine (500 mg, 2.15mmol) in a mixed solvent of dioxane (5 mL) and water (0.5 mL) was added(3-fluorophenyl)boronic acid (273 mg, 1.96 mmol), K₂CO₃ (811 mg, 5.88mmol) and Pd(dppf)₂Cl₂ (79 mg, 0.098 mmol). The reaction mixture wasstirred at 80° C. overnight under N₂. TLC (PE:EA=5:1) showed thestarting material was consumed. The mixture was cooled and concentrated.The residue was purified by column chromatography on silica gel(petroleum ether/EtOAc=20:1 to 1:1) to give6-chloro-3-(3-fluorophenyl)imidazo[1,2-b]pyridazine (300 mg, 56%) as ayellow solid.

To a solution of give6-chloro-3-(3-fluorophenyl)imidazo[1,2-b]pyridazine (100 mg, 0.404 mmol)in NMP (2 mL) was added 2-(trans-4-aminocyclohexyl)propan-2-ol (95 mg,0.6 mmol) and NaHCO₃ (101.8 mg, 1.21 mmol). The mixture was purged withnitrogen and kept at 180° C. for 30 min with M.W. reaction. LCMS showedthe reaction was complete. The mixture was partitioned between water(100 mL) and EtOAc (50 mL). The aqueous layer was extracted with EtOAc(50 mL*3). The combined extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by prep. HPLC to giveEX. 8-75 (10.6 mg, 7.2%) as a yellow solid.

¹H NMR (MeOD, 400 MHz): δ 8.26 (dt, J₁=11.34 Hz, J₂=2.06 Hz, 1H), 7.88(d, J=7.90 Hz, 1H), 7.86 (s, 1H), 7.63 (d, J=9.96 Hz, 1H), 7.48-7.43 (m,1H), 7.07 (td, J₁=8.42 Hz, J₂=2.75 Hz, 1H), 6.71 (d, J=9.62 Hz, 1H),3.68 (br, 1H), 2.36 (br, 2H), 2.00 (br, 2H), 1.42-1.26 (m, 5H), 1.21 (s,6H). MS (ES⁺, m/z): (M+H)⁺: 369.3

76.2-(trans-4-(((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)cyclohexyl)propan-2-ol(EX. 8-76)

To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (2 g,12.7 mmol) in MeOH (64 mL) was added dropwise thionyl chloride (0.93 mL,12.7 mmol) at room temperature. The resulting mixture was stirred atroom temperature for 4 h. The mixture was concentrated in vacuo todryness to give trans-methyl 4-(aminomethyl)cyclohexanecarboxylate (2.5g, 115%) as HCl salts.

To a solution of trans-methyl 4-(aminomethyl)cyclohexanecarboxylate (1g, 5.8 mmol) in THF (50 mL) was added dropwise methylmagnesium bromide(7.7 mL, 23.2 mmol) at −78° C. The mixture was stirred at roomtemperature for 2 h. TLC (PE:EA=5:1) showed the reaction was complete.The mixture was quenched by the addition of NH₄Cl (100 mL) and extractedwith EtOAc (100 mL×3). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo to give2-(trans-4-(aminomethyl)cyclohexyl)propan-2-ol (0.42 g, 39.2%) as ayellow solid.

A mixture of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (100mg, 0.38 mmol), 2-(trans-4-(aminomethyl)cyclohexyl)propan-2-ol (140 mg,0.76 mmol) and KF (51 mg, 0.88 mmol) in DMSO (2 mL) was stirred at 130°C. overnight. LCMS showed the reaction was complete. The mixture waspoured into water (100 mL) and extracted with EtOAc (50 mL×3). Thecombined extracts were dried over Na₂SO₄, filtered and concentrated invacuo. The residue was purified by prep-HPLC to give EX. 8-76 (12 mg,8%) as a yellow solid.

¹H NMR (MeOD, 400 MHz): δ 8.44 (s, 1H), 8.05 (s, 1H), 7.98 (d, J=7.56Hz, 1H), 7.78 (d, J=9.62 Hz, 1H), 7.50 (t, J=7.56 Hz, 1H), 7.43 (d,J=8.25 Hz, 1H), 6.97 (d, J=9.62 Hz, 1H), 3.27 (d, J=6.87 Hz, 2H), 2.03(d, J=10.65 Hz, 2H), 1.94 (s, J=10.65 Hz, 2H), 1.77 (br, 1H), 1.35-1.31(m, 1H), 1.15 (s, 6H), 1.15-1.02 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 399.3.

77.2-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)oxy)cyclohexyl)propan-2-ol(EX. 8-77)

To a solution of trans-ethyl 4-hydroxycyclohexanecarboxylate (1 g, 5.8mmol) in THF (50 mL) was added dropwise methylmagnesium bromide (7.7 mL,23.2 mmol) at −78° C. The mixture was stirred at room temperature for 2h. TLC (PE:EA=1:1) showed the reaction was complete. The mixture wasquenched by the addition of NH₄Cl (100 mL) and extracted with EtOAc (100mL*3). The combined extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo to give trans-4-(2-hydroxypropan-2-yl)cyclohexanol(0.88 g, 96%) as a yellow solid.

To a solution of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine(100 mg, 0.378 mmol) in toluene (2 mL) was addedtrans-4-(2-hydroxypropan-2-yl)cyclohexanol (179.8 mg, 1.14 mmol), BINAP(23.5 mg, 0.038 mmol), Pd(OAc)₂ (8.78 mg, 0.038 mmol) and t-BuONa (60.5mg, 0.76 mmol). The mixture was filled with nitrogen and kept 120° C.for 1 h with M.W. reaction. LCMS showed the reaction was complete. Themixture was poured into brine (100 mL) and extracted with EtOAc (100mL*3). The combined extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by prep. HPLC to givetrans-4-(2-hydroxypropan-2-yl)cyclohexanol (13 mg, 8.6%) as a yellowsolid.

¹H NMR (DMSO, 400 MHz): δ 8.41 (s, 1H), 8.18 (s, 1H), 8.05 (d, J=9.62Hz, 1H), 7.97 (d, J=7.56 Hz, 1H), 7.48 (t, J=7.56 Hz, 1H), 7.38 (d,J=7.56 Hz, 1H), 6.89 (d, J=9.88 Hz, 1H), 4.84-4.79 (m, 1H), 4.11 (s,1H), 2.28 (d, J=7.82 Hz, 2H), 1.88 (d, J=9.88 Hz, 2H), 1.44-1.36 (m,2H), 1.25-1.18 (m, 3H), 1.02 (s, 6H), MS (ES⁺, m/z): (M+H⁺): 386.2

78.trans-N1-(tert-Butyl)-N4-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(EX. 8-78)

To a solution of 1,4-dioxaspiro[4.5]decan-8-one (10 g, 6.4 mmol) andbenzylamine (7 mL, 6.4 mmol) in 1,2-dichloroethane (220 mL) was treatedwith NaBH(OAc)₃ (19 g, 89.6 mmol). The mixture was stirred at roomtemperature for 1 h. TLC (PE:EA=1:1) showed the reaction was complete.The mixture was partitioned between sat. aq. NaHCO₃ (500 mL) and EtOAc(200 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated in vacuo to give N-benzyl-1,4-dioxaspiro[4.5]decan-8-amine(18 g, 115%) as a yellow liquid, which was used in the next stepdirectly.

A mixture of N-benzyl-1,4-dioxaspiro[4.5]decan-8-amine (3 g, 12.2 mmol)and Palladium hydroxide (0.9 g) in MeOH (120 mL) was hydrogenated under50 psi of H₂ at room temperature. The mixture was stirred at roomtemperature for 24 h. TLC (CH₂Cl₂:MeOH=10:1) showed the reaction wascomplete. The mixture was filtered and the filtrated was concentrated invacuo to give 1,4-dioxaspiro[4.5]decan-8-amine (2.1 g, 110%) as a brownoil.

A mixture of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (500mg, 1.89 mmol), 4-dioxaspiro[4.5]decan-8-amine (297 mg, 1.89 mmol),BINAP (117.7 mg, 0.189 mmol), t-BuONa (302.4 mg, 3.78 mmol) andPd₂(dba)₃ in toluene (10 mL) was filled with N₂ and stirred at 110° C.overnight. TLC (PE:EA=1:1) showed the starting material was almostconsumed. The mixture was partitioned between water (150 mL) and EtOAc(100 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated in vucuo. The residue was purified by combi flash to give3-(3-chlorophenyl)-N-(1,4-dioxaspiro[4.5]decan-8-yl)imidazo[1,2-b]pyridazin-6-amine(420 mg, 57.8%) as a yellow solid.

To a solution of3-(3-chlorophenyl)-N-(1,4-dioxaspiro[4.5]decan-8-yl)imidazo[1,2-b]pyridazin-6-amine(210 mg, 0.55 mmol) in THF (3 mL) was added 3 N HCl (3 mL). Theresulting mixture was stirred at room temperature for 2 h. TLC (EtOAc)showed the reaction was complete. The mixture was poured into sat.NaHCO₃ (aq) and extracted with EtOAc (100 mL×3). The organic layers weredried over Na₂SO₄, filtered and concentrated in vacuo to give4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(190 mg, 102%) as a yellow solid.

To a 0° C. solution of4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(80 mg, 0.23 mmol) and t-Butylamine (17.2 mg, 0.23 mmol) in CH₂Cl₂ (1.2mL) was added dropwise Ti(Oi-Pr)₄ (0.27 mL, 0.92 mmol). The resultingmixture was stirred at room temperature overnight. LCMS showed thereaction was complete. The mixture was poured into water (150 mL) andextracted with EtOAc (100 mL*3). The organic layers were dried overNa₂SO₄, filtered and concentrated in vacuo. The residue was purified byprep. HPLC to give EX. 8-78 (15 mg, 16.5%) as a white solid.

¹H NMR (MeOD, 400 MHz): δ 8.56 (t, J=1.72 Hz, 1H), 8.45 (br, 0.66H),7.91 (d, J=7.93 Hz, 1H), 7.88 (s, 1H), 7.67 (d, J=9.66 Hz, 1H), 7.46 (t,J=7.93 Hz, 1H), 7.35 (dd, J₁=7.93 Hz, J₂=1.03 Hz, 1H), 6.75 (d, J=9.66Hz, 1H), 3.78 (tt, J₁=11.04 Hz, J₂=3.45 Hz, 1H), 3.42 (tt, J₁=12.07 Hz,J₂=3.79 Hz, 1H), 2.42 (d, J=11.04 Hz, 2H), 2.23 (d, J=12.42 Hz, 2H),1.79 (q, J=12.42 Hz, 2H), 1.54 (q, J=12.07 Hz, 2H), 1.46 (s, 9H), MS(ES⁺, m/z): (M+H⁺): 398.3.

79.N-(trans-4-(2-aminopropan-2-yl)cyclohexyl)-3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-amine(EX. 8-79)

To a solution of TMSN₃ (93 mg, 0.78 mmol), TsOH H₂O (49 mg, 0.26 mmol)and BF₃Et₂O (74 mg, 0.52 mmol) in CH₂Cl₂ (1.3 mL) was slowly addeddropwise a solution of EX. 8-37 (100 mg, 0.26 mmol) in CH₂Cl₂ (1 mL) at0° C. After the addition, the reaction mixture was stirred at roomtemperature overnight. TLC (CH₂Cl₂:MeOH=10:1) showed the reaction wascomplete. The mixture was poured into water (150 mL) and extracted withEtOAc (100 mL*3). The organic layers were dried over Na₂SO₄, filteredand concentrated in vacuo to give crudeN-(trans-4-(2-azidopropan-2-yl)cyclohexyl)-3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-amine(80 mg) as a yellow oil, which was used in the next step directly.

A mixture ofN-(trans-4-(2-azidopropan-2-yl)cyclohexyl)-3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-amine(90 mg, 0.22 mmol) and Pd/C (30 mg) in MeOH (5 mL) and CH₂Cl₂ (5 mL) washydrogenated under 50 psi of H₂ at room temperature for 48 h. LCMSshowed the reaction was complete. The mixture was filtered and thefiltrate was concentrated in vacuo. The residue was purified by prep.HPLC to give EX. 8-79 (4 mg, 5%) as an off-white solid.

¹H NMR (MeOD, 400 MHz): δ 8.49 (s, 1H), 8.29 (br, 0.68H), 7.79 (d,J=7.56 Hz, 1H), 7.77 (s, 1H), 7.56 (d, J=9.62 Hz, 1H), 7.34 (t, J=7.56Hz, 1H), 7.23 (d, J=7.56 Hz, 1H), 6.62 (d, J=9.62 Hz, 1H), 3.65 (tt,J₁=11.68 Hz, J₂=4.12 Hz, 1H), 2.29 (d, J=11.68 Hz, 2H), 1.81 (s, J=12.37Hz, 2H), 1.52 (t, J=11.68 Hz, 1H), 1.36 (q, J=11.68 Hz, 4H), 1.39-1.29(m, 4H), 1.25 (s, 6H). MS (ES⁺, m/z): (M+H⁺): 384.2.

80.N-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)methanesulfonamide(EX. 8-80)

To a mixture of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (500mg, 1.89 mmol) in toluene (10 mL) was addedtrans-cyclohexane-1,4-diamine (215.9 mg, 1.89 mmol), t-BuONa (302.4 mg,3.78 mmol), BINAP (117.7 mg, 0.189 mmol), Pd₂(dba)₃ (173 mg, 0.189mmol). The mixture was filled with N₂ and heated at 110° C. for 4 h. TLC(PE:EA=1:1) showed the reaction was complete. The reaction was pouredinto water (150 mL), extracted with EA (3×50 mL). The organic layer wasdried and condensed. The residue was purified by combi flash(CH₂Cl₂:MeOH=0˜10:1) to givetrans-N1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(240 mg, yield: 37%) as a yellow solid.

To a 0° C. solution oftrans-N1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(50 mg, 0.146 mmol) and DIPEA (0.08 mL, 0.44 mmol) in DMSO (0.6 mL) wasadded dropwise MeSO₃Cl (20 mg, 0.176 mmol). The resulting mixture wasstirred at room temperature for 6 h. TLC (CH₂Cl₂:MeOH=10:1) showed mostof starting material was converted. The mixture was partitioned betweenwater (50 mL) and EtOAc (50 mL) and separated. The aqueous layer wasextracted with EtOAc (50 mL×3). The combined extracts were dried overNa₂SO₄, filtered and concentrated in vacuo. The residue was purified byprep-HPLC to give EX. 8-80 (10.8 mg, 18%) as a white solid.

¹H NMR (DMSO, 400 MHz): δ 8.53 (s, 1H), 8.23 (br, 0.4H), 8.01 (d, J=8.0Hz, 1H), 7.99 (s, 1H), 7.75 (d, J=9.6 Hz, 1H), 7.45 (t, J=8.0 Hz, 1H),7.35 (dd, J₁=7.6 Hz, J₂=1.2 Hz, 1H), 7.10 (d, J=7.6 Hz, 1H), 7.07 (d,J=6.4 Hz, 1H), 6.69 (d, J=9.6 Hz, 1H), 3.54-3.51 (m, 1H), 3.21-3.15 (m,1H), 2.92 (s, 3H), 2.17 (d, J=10.4 Hz, 2H), 1.98 (d, J=10.4 Hz, 2H),1.46-1.30 (m, 4H). MS (ES⁺, m/z): (M+H⁺): 420.2

81.4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexane-1-sulfonamide(EX. 8-81)

To a solution of isoindoline-1,3-dione (2 g, 13.6 mmol) in Et₃N (60 mL)was added ClCOOEt (2.55 g, 16.33 mmol) at 0° C. The mixture was warmedto room temperature and stirred at room temperature for 4 h. The mixturewas filtered and the solid was partitioned between water (150 mL) andEtOAc (50 mL) and adjusted pH value to 6 with 1 N HCl. The organic layerwas washed with brine and dried over Na₂SO₄, filtered and concentratedin vacuo to give ethyl 1,3-dioxoisoindoline-2-carboxylate (1.67 g,53.08%) as a white solid.

To a solution of ethyl 1,3-dioxoisoindoline-2-carboxylate (1.67 g, 7.19mmol) in water (16 mL) was added 4-aminocyclohexanol (1.31 g, 8.63mmol), followed by K₂CO₃ (1.59 g, 11.5 mmol). The mixture was stirred atroom temperature for 2 h. TLC (PE:EA=1:1) showed the reaction wascomplete. The mixture was filtered and the solid was dried under vacuumto give 2-(4-hydroxycyclohexyl)isoindoline-1,3-dione (1 g, 56.82%) as abrown solid.

To a solution of PPh₃ (1.28 g, 4.9 mmol) in THF (12 mL) was addeddropwise DEAD (852 mg, 4.9 mmol) at −10° C. After the white solid wasappeared, compound 2-(4-hydroxycyclohexyl)isoindoline-1,3-dione (600 mg,2.45 mmol) was added, followed by CH₃COSH (0.35 mL, 4.9 mmol) at −10° C.The mixture was stirred at room temperature overnight. TLC (PE:EA=1:1)showed the reaction was complete. The mixture was poured into water (200mL) and extracted with EtOAc (100 mL). The aqueous layer was extractedwith EtOAc (100 mL×3). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo to giveS-(4-(1,3-dioxoisoindolin-2-yl)cyclohexyl) ethanethioate (600 mg, 81%)as a white solid.

A solution of hydrogen peroxide (0.8 mL) in formic acid (8 mL) wasstirred at room temperature for 1 h. After cooling in an ice bath, amixture of compound 60 (400 mg, 1.32 mmol) in formic acid (2 mL) wasadded while keeping the temperature at 0° C. The mixture was stirred atroom temperature overnight. TLC (PE:EA=1:1) showed the reaction wascomplete. The mixture was cooled in an ice bath and solid Na₂SO₃ wasadded slowly till the iodine-starch did not go blue. The mixture wasmixed with silica gel and concentrated in vacuo. The residue waspurified by combi flash (CH₂Cl₂:MeOH=10:1) to give4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonic acid (400 mg, 98%) asa white solid.

A mixture of 4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonic acid(6.8 g, 22 mmol) and PCl₅ (5 g, 24 mmol) was stirred at room temperaturefor 30 min. And then the mixture was heated at 100° C. and stirred for 2h. After cooling to room temperature, the mixture was washed with EtOAc(150 mL×3). The combined extracts were concentrated in vacuo to givecrude 4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonyl chloride (5 g,68%) as a whit solid, which was used in the next step directly.

A mixture of 4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonyl chloride(5 g, 15.2 mmol) in NH₃/THF (150 mL) was stirred at room temperatureovernight. The mixture was concentrated in vacuo. The residue wasdissolved into about 100 mL of hot water and the solid was filteredunder hot condition. The filtrate was concentrated in vacuo to give4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonamide (3 g, 64%) as anoff-white solid.

A mixture of 4-(1,3-dioxoisoindolin-2-yl)cyclohexane-1-sulfonamide (3 g,9.7 mmol) and NH₂NH₂H₂O (1 mL) in EtOH (15 mL) was heated at reflux for30 min. After cooling, the pH was adjusted with diluted HCl to 5. Themixture was concentrated in vacuo. The residue was dissipated with 35%HCl (10 mL) and heating until all solid material was dissolved. Uponcooling, a crystalline material was filtered and the filtrated wasconcentrated to give crude 4-aminocyclohexane-1-sulfonamide (700 mg,40%) as a yellow solid, which was used in the next step directly.

A mixture of 4-aminocyclohexane-1-sulfonamide (200 mg, 1.12 mmol),6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (200 mg, 0.76 mmol)and KF (500 mg, 8.6 mmol) in DMSO (2 mL) was heated at 120° C.overnight. LCMS showed the reaction was complete. The mixture was pouredinto brine (80 mL) and extracted with EtOAc (30 mL×3). The combinedextracts were dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by to prep. HPLC give two isomers: EX. 8-81 isomer1 (7.0 mg) and EX. 8-81 isomer 2 (8.1 mg) as white solid.

EX. 8-81 isomer 1. ¹H NMR (MeOD, 400 MHz): δ 8.39 (s, 1H), 8.33 (s, 1H),8.00-7.97 (m, 2H), 7.58-7.55 (m, 2H), 7.25 (d, J=9.93 Hz, 1H), 3.78 (tt,J₁=11.35 Hz, J₂=3.41 Hz, 1H), 3.02 (tt, J₁=11.64 Hz, J₂=3.41 Hz, 1H),2.39 (t, J=11.64 Hz, 4H), 1.79 (d, J=12.49 Hz, 2H), 1.47 (d, J=12.49 Hz,2H). MS (ES⁺, m/z): (M+H⁺): 406.1.

EX. 8-81 isomer 2. ¹H NMR (MeOD, 400 MHz): δ 8.34 (s, 1H), 8.32 (s, 1H),8.0 (d, J=9.96 Hz, 1H), 7.97 (d, J=6.87 Hz, 1H), 7.56 (d, J=7.56 Hz,1H), 7.55 (s, 1H), 7.40 (d, J=9.96 Hz, 1H), 4.08 (t, J=3.44 Hz, 1H),3.09 (tt, J₁=9.96 Hz, J₂=3.78 Hz, 1H), 2.29-2.25 (m, 2H), 2.15-2.10 (m,2H), 2.06-1.96 (m, 2H), 1.88-1.80 (m, 2H). MS (ES⁺, m/z): (M+H⁺): 406.2.

82.2-(trans-3-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclopentyl)propan-2-ol(EX. 8-82)

To a 80° C. mixture of trans-3-aminocyclopentanecarboxylic acid (1 g,7.74 mmol) and K₂CO₃ (3.2 g, 23.22 mmol) in CH₃CN (20 mL) was addeddropwise a solution of benzyl bromide (3.2 mL, 27.1 mmol) in CH₃CN (10mL). The mixture was heated at 80° C. overnight. The mixture was cooledand poured into water (150 mL) and extracted with EtOAc (100 mL*3). Thecombined extracts were dried over Na₂SO₄, filtered and concentrated invacuo. The residue was purified by combi flash to give trans-benzyl3-(dibenzylamino)cyclopentanecarboxylate (2.8 g, 90.6%) as a whitesolid.

To a 0° C. solution of trans-benzyl3-(dibenzylamino)cyclopentanecarboxylate (2.8 g, 7 mmol) in THF (250 mL)was added dropwise MeMgBr (23 mL, 70 mmol). After the addition, thereaction mixture was stirred at room temperature overnight. TLC (PE)showed the reaction was complete. The mixture was cooled and poured intosat. NH₄Cl (300 mL) and extracted with EtOAc (100 mL*3). The combinedextracts were dried over Na₂SO₄, filtered and concentrated in vacuo togive crude 2-(trans-3-(dibenzylamino)cyclopentyl)propan-2-ol (3 g, 133%)as a yellow liquid.

A mixture of 2-(trans-3-(dibenzylamino)cyclopentyl)propan-2-ol (3 g, 9.3mmol) and Pd(OH)₂ (900 mg) in EtOH (30 mL) was hydrogenated under 50 psiof H₂ for 48 h at room temperature. TLC (PE:EA=5:1) showed the reactionwas complete. The mixture was filtered and concentrated in vacuo to givecrude 2-(trans-3-aminocyclopentyl)propan-2-ol (1 g, 77%) as a yellowoil.

A mixture of 2-(trans-3-aminocyclopentyl)propan-2-ol (82 mg, 0.57 mmol),6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (100 mg, 0.38 mmol)and KF (66.2 mg, 1.14 mmol) in DMSO (1 mL) was stirred at 130° C.overnight. LCMS showed the reaction was complete. The mixture was pouredinto water (100 mL) and extracted with EtOAc (50 mL*3). The combinedextracts were dried over Na₂SO₄, filtered and concentrated in vacuo. Theresidue was purified by prep-HPLC to give EX. 8-82 (45 mg, 32%) as anoff-white solid.

¹H NMR (MeOD, 400 MHz): δ 8.57 (t, J=2.06 Hz, 1H), 7.96 (dt, J₁=8.11 Hz,J₂=1.32 Hz, 1H), 7.86 (s, 1H), 7.63 (d, J=9.88 Hz, 1H), 7.44 (t, J=8.11Hz, 1H), 7.33 (d, J=7.76 Hz, 1H), 6.74 (d, J=9.70 Hz, 1H), 4.19-4.12 (m,1H), 2.41-2.34 (m, 1H), 2.22-2.10 (m, 2H), 1.83-1.62 (m, 3H), 1.51-1.43(m, 1H), 1.23 (s, 6H). MS (ES⁺, m/z): (M+H⁺): 371.1

83.N-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)formamide(EX. 8-83)

A mixture oftrans-N1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(50 mg, 0.146 mmol) and ethyl formate (0.37 mL) in DMF (1.2 mL) washeated at 90° C. for 6 h. LCMS showed the reaction was complete. Themixture was concentrated in vacuo and purified b prep-HPLC to give EX.8-83 (22 mg, 41%) as an off-white solid.

¹H NMR (MeOD, 400 MHz): δ 8.47 (s, 1H), 8.23 (br, 0.36H), 7.98 (s, 1H),7.88 (d, J=8.0 Hz, 1H), 7.82 (s, 1H), 7.60 (d, J=9.6 Hz, 1H), 7.39 (t,J=8.0 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.68 (d, J=9.6 Hz, 1H), 3.79 (t,J=11.2 Hz, 1H), 3.70 (t, J=10.8 Hz, 1H), 2.28 (d, J=12 Hz, 2H), 2.02 (d,J=12 Hz, 2H), 1.54-1.36 (m, 4H). MS (ES⁺, m/z): (M+H⁺): 370.2

84.N-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)-2-morpholinoacetamide(EX. 8-84)

To a solution oftrans-N1-(3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(90 mg, 0.26 mmol) and pyridine (0.08 mL, 1.04 mmol) in CH₂Cl₂ (2.6 mL)was added dropwise chloroacetyl chloride (0.03 mL, 0.39 mmol). Theresulting mixture was stirred at room temperature overnight. TLC(CH₂Cl₂:MeOH=10:1) showed the reaction was complete. The mixture waspoured into sat. aq. NaHCO₃ (100 mL) and extracted with EtOAc (50 mL*3).The extracts were dried over Na₂SO₄, filtered and concentrated in vacuoto give crude2-chloro-N-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)acetamide(110 mg, 101%) as a yellow solid.

A mixture of2-chloro-N-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)acetamide(50 mg, 0.12 mmol), morpholine (0.3 mL) and DIPEA (0.2 mL) in CH₂Cl₂(0.6 mL) was stirred at 50° C. for 3 h. LCMS showed the reaction wascomplete. The mixture was concentrated in vacuo. The residue waspartitioned between water (100 mL) and EtOAc (50 mL). The organic layerwas dried over Na₂SO₄, filtered and concentrated in vacuo. The residuewas purified by prep-HPLC to give EX. 8-84 (9.6 mg, 17%) as a whitesolid.

¹H NMR (MeOD, 400 MHz): δ 8.49 (s, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.88 (s,1H), 7.66 (d, J=9.6 Hz, 1H), 7.44 (t, J=7.6 Hz, 1H), 7.34 (d, J=8.8 Hz,1H), 6.75 (d, J=9.6 Hz, 1H), 3.87-3.73 (m, 6H), 3.36-3.30 (m, 1H),3.36-3.30 (m, 1H), 3.30-3.20 (m, 1H), 2.84 (br, 4H), 2.31 (d, J=12.8 Hz,2H), 2.02 (d, J=12.8 Hz, 2H), 1.54-1.40 (m, 4H). MS (ES⁺, m/z): (M+H⁺):469.3.

85.trans-4-((3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(EX. 8-85)

A mixture of6-chloro-3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazine (50 mg,0.168 mmol), Trans-4-aminocyclohexanecarbonitrile (53 mg, 0.22 mmol) andKF (42.2 mg, 0.73 mmol) in DMSO (0.5 mL) was heated at 130° C.overnight. LCMS showed the reaction was almost complete. The mixture waspurified by prep. HPLC to givetrans-4-((3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(20 mg, 30.8%) as an off-white solid.

¹H NMR (MeOD, 400 MHz): δ 8.75 (s, 1H), 8.24 (t, J=3.61 Hz, 1H), 7.99(s, 1H), 7.74-7.69 (m, 3H), 6.84 (d, J=9.79 Hz, 1H), 3.83 (tt, J₁=10.82Hz, J₂=3.78 Hz, 1H), 2.71 (tt, J₁=11.51 Hz, J₂=3.61 Hz, 1H), 2.23 (tt,J₁=16.15 Hz, J₂=6.18 Hz, 4H), 1.73 (q, J=12.20 Hz, 2H), 1.43 (q, J=12.54Hz, 2H). MS (ES⁺, m/z): (M+H⁺):386.2.

86.trans-4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(EX. 8-86)

EX. 8-86 is prepared from6-chloro-3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazine usingsimilar procedures as in EX. 8-85.

¹H NMR (MeOD, 400 MHz): δ 8.42 (s, 1H), 8.02 (d, J=7.90 Hz, 1H), 7.99(s, 1H), 7.72 (d, J=9.62 Hz, 1H), 7.60 (t, J=8.25 Hz, 1H), 7.31 (t,J=8.25 Hz, 1H), 6.82 (t, J=9.62 Hz, 1H), 3.82 (tt, J₁=10.65 Hz, J₂=3.44Hz, 1H), 2.73 (tt, J₁=11.68 Hz, J₂=3.44 Hz, 1H), 2.31-2.20 (m, 4H), 1.76(q, J=12.02 Hz, 2H), 1.43 (q, J=12.02 Hz, 2H). MS (ES⁺, m/z): (M+H⁺):402.2.

87.2-(trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)acetonitrile(EX. 8-87)

To a 0° C. solution of diethyl cyanomethyl phosphonate (0.07 mL, 0.4mmol) in THF (1.5 mL) was added DMPU (0.27 mL, 1 mmol), followed by NaH(15 mL, 0.37 mmol). After stirring for 10 min, a solution of4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(130 mg, 0.38 mmol) in THF (1.5 mL) was added. The resulting mixture wasstirred at room temperature for 2 h. TLC (EA) showed most of s.m. wasconverted into product. The mixture was poured into water (100 mL) andextracted with EtOAc (50 mL*3). The combined extracts were dried overNa₂SO₄, filtered and concentrated in vacuo to give crude2-(4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexylidene)acetonitrile(100 mg, 69%) as a yellow solid.

A mixture of2-(4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexylidene)acetonitrile(100 mg, 0.26 mmol) and 10% Pd/C (100 mg) in EtOAc (2 mL) was stirredunder H₂ balloon at room temperature for 4 h. LCMS showed the most ofs.m was consumed. The mixture was filtered and the filterate wasconcentrated in vacuo. The residue was purified by prep. HPLC to giveEX. 8-87 (13 mg, 13.7%) as a white solid.

¹H NMR (MeOD, 400 MHz): δ 8.51 (s, 1H), 8.17 (br, 0.4H), 7.93 (d, J=7.90Hz, 1H), 7.87 (s, 1H), 7.64 (d, J=9.96 Hz, 1H), 7.44 (t, J=7.90 Hz, 1H),7.34 (d, J=7.90 Hz, 1H), 6.73 (d, J=9.96 Hz, 1H), 3.72 (tt, J₁=10.31 Hz,J₂=3.44 Hz, 1H), 2.46 (d, J=6.18 Hz, 2H), 2.33 (d, J=10.65 Hz, 2H), 1.98(d, J=10.99 Hz, 2H), 1.77 (br, 1H), 1.48-1.32 (m, 4H). MS (ES⁺, m/z):(M+H⁺): 366.3.

88.trans-4-(((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)methyl)cyclohexanecarbonitrile(EX. 8-88)

To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (2 g,12.7 mmol) in MeOH (64 mL) was added dropwise thionyl chloride (0.927mL, 12.7 mmol) at room temperature. The resulting mixture was stirred atroom temperature for 4 h. The mixture was concentrated in vacuo to givecrude trans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexanecarboxylicacid (2.5 g, 115%) as HCl salts, which was used in the next stepdirectly.

A suspension oftrans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexanecarboxylic acid(2.27 g, 8.8 mmol) and NMM (1.4 mL, 10.12 mmol) in THF (15 mL) was addeddropwise isopropyl chloroformate (1.79 g, 17.6 mmol) below 10° C. Themixture containingtrans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexanecarboxylic(isopropyl carbonic) anhydride was stirred at room temperature for 3 h.TLC (PE:EA=1:1) showed the reaction was complete. The mixture was usedin the next step directly.

Above mixture was cooled to 10° C. again and ammonium hydroxide (8.2 mL,52.8 mmol) was added. The final mixture was stirred at room temperaturefor 18 h. The mixture was filtered and the filter cake was washed withwater three times and dried to givetert-butyl((trans-4-carbamoylcyclohexyl)methyl)carbamate (1.7 g, 74%) asa white solid.

To a solution oftert-butyl((trans-4-carbamoylcyclohexyl)methyl)carbamate (1.7 g, 6.6mmol) in CH₂Cl₂ (33 mL) was added DBU (3 g, 19.8 mmol) at roomtemperature. After stirring for 10 min, ethyl dichlorophosphate (2.2 g,13.2 mmol) was added. The mixture was stirred at room temperature for 50min. TLC (CH₂Cl₂:MeOH=10:1) showed the reaction was complete. Themixture was poured into sat. NH₄Cl (aq., 150 mL) and extracted withCH₂Cl₂ (100 mL*2). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified by combiflash to give tert-butyl((trans-4-cyanocyclohexyl)methyl)carbamate (1.4g, 88%) as a white solid.

To a solution of tert-butyl((trans-4-cyanocyclohexyl)methyl)carbamate(1.4 g, 5.86 mmol) in CH₂Cl₂ (20 mL) was added TFA (20 mL) at r.t. Theresulting mixture was stirred at room temperature for 1 h. TLC(PE:EA=5:1) showed the reaction was complete. The mixture wasconcentrated in vacuo to givetrans-4-(aminomethyl)cyclohexanecarbonitrile (2.6 g, 319%) as TFA salts.

A mixture of 6-chloro-3-(3-chlorophenyl)imidazo[1,2-b]pyridazine (50 mg,0.189 mmol), -4-(aminomethyl)cyclohexanecarbonitrile (111 mg, 0.25 mmol)and KF (47 mg, 0.82 mmol) in DMSO (0.5 mL) was heated at 130° C.overnight. LCMS showed the reaction was almost complete. The mixture waspurified by prep. HPLC to give EX. 8-88 (26 mg, 38%) as an off-whitesolid.

¹H NMR (MeOD, 400 MHz): δ 8.48 (t, J=1.89 Hz, 1H), 8.19 (br, 0.3H), 7.95(dt, J₁=7.90 Hz, J₂=1.37 Hz, 1H), 7.86 (s, 1H), 7.66 (d, J=9.62 Hz, 1H),7.45 (t, J=7.90 Hz, 1H), 7.35 (dq, J₁=8.07 Hz, J₂=0.86 Hz, 1H), 6.76 (d,J=9.62 Hz, 1H), 3.28 (d, J=6.87 Hz, 2H), 2.60 (tt, J₁=12.02 Hz, J₂=3.61Hz, 1H), 2.17-2.13 (m, 2H), 2.05-2.01 (m, 2H), 1.94-1.87 (m, 1H), 1.59(qd, J₁=13.06 Hz, J₂=3.26 Hz, 1H), 1.14 (qd, J₁=12.71 Hz, J₂=3.26 Hz,1H). MS (ES⁺, m/z): (M+H⁺): 366.1.

EX. 8-89, Ex. 8-90 and EX. 8-91 were prepared using similar proceduresas in Ex. 8-80.

89.N-(trans-4-((3-(3-fluorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)methanesulfonamide(EX. 8-89)

To a 0° C. solution oftrans-N1-(3-(3-fluorophenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(180 mg, 0.55 mmol) and DIPEA (213.2 mg, 1.65 mol) in DMSO (2.8 mL) wasadded dropwise MeSO₂Cl (63.4 mg, 0.55 mmol). The resulting mixture wasstirred at room temperature overnight. LCMS showed most of startingmaterial was converted. The mixture was partitioned between brine (100mL) and EtOAc (50 mL) and separated. The aqueous layer was extractedwith EtOAc (50 mL*3). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified byprep-HPLC to give EX. 8-89 (13 mg, 6%) as a brown solid.

¹H NMR (MeOD, 400 MHz): δ 8.13 (dt, J₁=10.65 Hz, J₂=2.06 Hz, 1H), 8.07(s, 1H), 7.89 (d, J=7.90 Hz, 1H), 7.80 (d, J=9.62 Hz, 1H), 7.53 (q,J=7.90 Hz, 1H), 7.17 (td, J₁=8.25 Hz, J₂=2.40 Hz, 1H), 6.96 (d, J=9.62Hz, 1H), 3.70 (tt, J₁=10.65 Hz, J₂=3.78 Hz, 1H), 3.32-3.27 (m, 1H), 2.99(s, 3H), 2.30 (d, J=12.02 Hz, 2H), 2.15 (d, J=11.68 Hz, 2H), 1.58-1.40(m, 4H). MS (ES⁺, m/z): (M+H⁺): 404.0.

90.N-(trans-4-((3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)methanesulfonamide(EX. 8-90)

To a 0° C. solution oftrans-N1-(3-(3-(trifluoromethyl)phenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(130 mg, 0.35 mmol) and DIPEA (135.7 mg, 10.5 mmol) in DMSO (1.75 mL)was added dropwise MeSO₂Cl (47.6 mg, 0.42 mmol). The resulting mixturewas stirred at room temperature overnight. LCMS showed most of startingmaterial was converted. The mixture was partitioned between brine (100mL) and EtOAc (50 mL) and separated. The aqueous layer was extractedwith EtOAc (50 mL*3). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified byprep-HPLC to give EX. 8-90 (30 mg, 18.9%) as a white solid.

¹H NMR (MeOD, 400 MHz): δ 8.73 (s, 1H), 8.28 (d, J=6.87 Hz, 1H), 7.93(s, 1H), 7.69-7.67 (m, 3H), 6.77 (d, J=9.62 Hz, 1H), 3.74 (tt, J₁=10.65Hz, J₂=3.78 Hz, 1H), 3.29-3.26 (m, 1H), 2.99 (s, 3H), 2.28 (d, J=11.68Hz, 2H), 2.12 (d, J=12.35 Hz, 2H), 1.56-1.39 (m, 4H). MS (ES⁺, m/z):(M+H⁺): 454.7.

91.N-(trans-4-((3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexyl)methanesulfonamide(EX. 8-91)

To a 0° C. solution oftrans-N1-(3-(3-(trifluoromethoxy)phenyl)imidazo[1,2-b]pyridazin-6-yl)cyclohexane-1,4-diamine(180 mg, 0.46 mmol) and DIPEA (178 mg, 1.38 mol) in DMSO (2.0 mL) wasadded dropwise MeSO₂Cl (53 mg, 0.46 mmol). The resulting mixture wasstirred at room temperature overnight. LCMS showed most of startingmaterial was converted. The mixture was partitioned between brine (100mL) and EtOAc (50 mL) and separated. The aqueous layer was extractedwith EtOAc (50 mL*3). The combined extracts were dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was purified byprep-HPLC to give EX. 8-91 (57.1 mg, 26.4%) as a white solid.

¹H NMR (MeOD, 400 MHz): δ 8.33 (s, 1H), 8.07 (d, J=8.25 Hz, 1H), 7.88(s, 1H), 7.65 (d, J=9.62 Hz, 1H), 7.56 (t, J=7.56 Hz, 1H), 7.27 (d,J=8.25 Hz, 1H), 6.73 (d, J=9.62 Hz, 1H), 3.73 (tt, J₁=10.99 Hz, J₂=3.44Hz, 1H), 3.32-3.28 (m, 1H), 2.99 (s, 3H), 2.29 (d, J=12.37 Hz, 2H), 2.14(d, J=11.68 Hz, 2H), 1.57-1.38 (m, 4H). MS (ES⁺, m/z): (M+H⁺): 470.2.

92.4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanoneoxime (EX. 8-92)

To a mixture of hydroxylamine hydrochloride (15.3 mg, 0.22 mmol) andNa₂CO₃ (28 mg, 0.26 mmol) in water (0.7 mL) was added a solution of4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanone(50 mg, 0.147 mmol) in MeOH (0.5 mL) at 0° C. The resulting mixture wasstirred at room temperature for 1 h. LCMS showed the reaction wascomplete. The mixture was poured into water and extracted with EtOAc (50mL*3). The combined extracts were dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by prep-HPLC and then bySFC to give EX. 8-92 (10 mg, 19%) as a white solid.

¹H NMR (MeOD, 400 MHz): δ 8.39 (s, 1H), 7.83 (d, J=8.25 Hz, 1H), 7.74(s, 1H), 7.54 (d, J=9.96 Hz, 1H), 7.34 (t, J=7.90 Hz, 1H), 7.23 (dd,J₁=7.99 Hz, J₂=1.12 Hz, 1H), 6.64 (d, J=9.71 Hz, 1H), 3.91 (tt, J₁=10.31Hz, J₂=3.44 Hz, 1H), 3.17-3.15 (m, 1H), 2.40-2.35 (m, 1H), 2.29-2.19 (m,3H), 2.07-2.00 (m, 2H), 1.51-1.36 (m, 1H). MS (ES⁺, m/z): (M+H⁺): 356.1.

93.trans-4-((3-(3-chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarboximidamide(EX. 8-93)

trans-4-((3-(3-Chlorophenyl)imidazo[1,2-b]pyridazin-6-yl)amino)cyclohexanecarbonitrile(100 mg, 0.252 mmol) was dissolved into HCl/MeOH (4 mL) and HCl/dioxane(4 mL). The mixture was stirred at room temperature overnight. Themixture was concentrated in vacuo and the residue was dissolved inNH₃/MeOH (4 mL). The mixture was stirred at room temperature for 2 h.LCMS showed the reaction was complete. The mixture was concentrated invacuo. The residue was purified by prep. HPLC to give EX. 8-93 (3.5 mg,18.4%) as a brown solid.

¹H NMR (MeOD, 400 MHz): δ 8.42 (br, 1H), 8.26 (s, 1H), 7.85 (d, J=8.25Hz, 1H), 7.72 (s, 1H), 7.55 (d, J=9.62 Hz, 1H), 7.35 (t, J=8.25 Hz, 1H),7.23 (d, J=8.25 Hz, 1H), 6.62 (d, J=9.62 Hz, 1H), 3.66 (tt, J₁=11.68 Hz,J₂=3.44 Hz, 1H), 2.47 (d, J₁=13.06 Hz, J₂=3.44 Hz, 1H), 2.36 (d, J=10.31Hz, 2H), 1.98 (d, J=12.37 Hz, 2H), 1.72 (qd, J₁=13.06 Hz, J₂=2.40 Hz,),1.32 (qd, J₁=12.71 Hz, J₂=2.75 Hz,).

MS (ES⁺, m/z): (M+H⁺): 369.1; (M/2+H⁺): 185.4.

95.trans-N1-(3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)cyclohexane-1,4-diamine(EX. 8-95)

EX. 8-95 was prepared using a similar procedures as in EX. 8-96.

96.(S)-1-(3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-amine(EX. 8-96)

A mixture of 5-chloro-3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidine (100mg, 0.379 mmol), (S)-piperidin-3-amine (76 mg, 0.757 mmol), and DIEA(0.158 ml, 1.515 mmol) in 2-propanol (3 ml) was irradiated to 150° C.for 14 h in a Biotage microwave. After cooling, the mixture was dilutedwith saturated aqueous NaHCO₃, and extracted three times with EtOAc. Thecombined extracts were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. After absorbing on celite, the compound waspurified by chromatography (12 g silica, 0-15% methanol/DCM) to give(S)-1-(3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-amine(35 mg, 28% yield) as a white solid.

¹H NMR (CD₃OD/400 MHz): 8.47 (d, J=8.0 Hz, 1H), 8.29 (s, 1H), 8.03 (s,1H), 7.92 (m, 1H), 7.33 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 6.73(d, J=8.0 Hz, 1H), 4.27 (m, 1H), 4.02 (m, 1H), 3.50 (m, 1H), 3.40 (m,1H), 2.20 (m, 1H), 1.90 (m, 1H), 1.80 (m, 2H), 1.36 (m, 1H). MS (ES⁺,m/z): (M+H)⁺: 328.5.

97.4-((3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexanone(EX. 8-97)

A mixture of 5-chloro-3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidine (100mg, 0.379 mmol), 4-aminocyclohexanone (43 mg, 0.379 mmol), and DIEA(0.158 ml, 1.515 mmol) in 2-propanol (3 ml) was irradiated to 150° C.for 14 h in a Biotage microwave. After cooling, the mixture was dilutedwith saturated aqueous NaHCO₃, and extracted three times with EtOAc. Thecombined extracts were washed with brine, dried over Na₂SO₄ andconcentrated in vacuo. After absorbing on celite, the compound waspurified by chromatography (12 g silica, 0-15% methanol/DCM) to give4-((3-(3-chlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexanone(30 mg, 24% yield) as a white solid.

¹H NMR (CD₃OD/400 MHz): 8.35 (s, 1H), 8.26 (d, J=7.6 Hz, 1H), 8.21 (d,J=10.4 Hz, 1H), 7.79 (m, 1H), 7.08 (m, 1H), 6.26 (m, 1H), 4.40 (m, 1H),2.62 (m, 2H), 2.47 (m, 2H), 2.00 (m, 1H), 1.84 (m, 2H), 1.65 (m, 1H). MS(ES⁺, m/z): (M+H)⁺: 341.4.

98.4-trans-((7-amino-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexyl)propan-2-ol(EX. 8-98)

A mixture of 3-trifluoromethoxyphenylacetonitrile (2.5 grams, 12.43mmol), DIPEA (0.321 grams, 2.48 mmol), and dimethyl formamide dimethylacetal (20 mL) were heated at reflux for 4 h. On cooling, the reactionmixture was partitioned between EtOAc and saturated aqueous NH₄Clsolution. The aqueous phase was extracted with ethyl acetate and thecombined organic phase was washed with brine and concentrated in vacuo.The crude product was purified by chromatography (ethyl acetate/hexane,0-10%) on silica gel (24 grams) to give pure product (2.3 grams, 8.98mmol, 72% yield.).

Synthesis of 4-(3-(trifluoromethoxy)phenyl)-1H-pyrazol-5-amine

A mixture of acrylnitrile (2.0 g, 7.81 mmol), hydrazine hydrate (4.53grams, 39.0 mmol), and glacial acetaic acid (2.34 grams, 39.0 mmol) andethanol (20 mL) were heated at reflux for 16 h. On cooling, the reactionmixture was diluted with water, extracted with AcOEt and the combinedorganic phase was washed with brine and concentrated in vacuo (1.90grams, 7.84 mmol, 100% yield.). ¹H-NMR (CDCl₃/400 MHz): δ 7.64 (m, 1H),7.42 (m, 4H), 7.06 (d, J=7.6 Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 244.3.

A mixture of pyrazole (2.0 g, 8.22 mmol), diethyl malonate (1.32 g, 8.22mmol), and dry EtOH 20 mL were treated dropwise with sodium ethoxide(1.12 g, 16.45 mmol) and on completion of addition the reaction washeated at reflux for 16 h. On cooling the reaction mixture wasconcentrated in vacuo and the residue was added to ice, neutralized withacetic acid and the resulting precipitate filtrated, washed with waterand dried to give product3-(3-trifluoromethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5,7-diol (1.6 g,5.14 mmol, 63% yield).

A mixture of3-(3-trifluoromethoxyphenyl)pyrazolo[1,5-a]pyrimidine-5,7-diol (1.0 g,3.21 mmol) in 20 mL phosphoryl oxychloride was heated at reflux for 16h. On cooling the reaction mixture was concentrated in vacuo and theresidue was extracted with ethyl ether three times. The combined ethersolution was washed with NaHCO₃ and dried. After removed the solvent,pure product5,7-dichloro-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidine wasobtained (0.8 g, 2.30 mmol, 72% yield).

To a microwave reactor charged with the 5,7-dichloro product (1.0 g,2.87 mmoL) and a stirbar was added saturated aqueous NH₃ 5 mL. Themicrowave was sealed and heated to 80° C. for 4 h. After cooled down andextracted with ethyl acetate, the organic phase was dried andconcentrated. Chromatography provided pure product5-chloro-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-7-amine(0.15 g, 0.456 mmol, 16% yield).

¹H-NMR (CD₃Cl/400 MHz): δ 8.32 (s, 1H), 7.95 (d, J=7.6 Hz, 1H), 7.83 (s,1H), 7.43 (t, J=8.0 Hz, 1H), 7.07 (d, J=7.6 Hz, 1H), 6.16 (s, 1H), 5.78(br, 2H). MS (ES⁺, m/z): (M+H)⁺: 329.5.

To a solution of5-chloro-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-7-amine(150 mg, 0.46 mmol) and 4-trans-(aminocyclohexyl)propan-2-ol (144 mg,0.913 mmol) in NMP (2.0 mL) was added NaHCO₃ (43 mg, 0.55 mmol), themixture was stirred at 180° C. for 60 mins under microwave irradiation.The mixture was purified by flash chromatograph to give4-trans-((7-amino-3-(3-(trifluoromethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)cyclohexyl)propan-2-ol(30 mg, 0.067 mmol, 15%) as a pale yellow solid.

¹H-NMR (CD₃OD/400 MHz): δ 8.40 (s, 1H), 8.20 (s, 1H), 7.78 (d, J=7.6 Hz,1H), 7.35 (t, J=8.0 Hz, 1H), 6.94 (d, J=7.6 Hz, 1H), 5.38 (s, 1H), 3.84(m, 1H), 3.41 (m, 1H), 2.20 (m, 2H), 1.95 (m, 2H), 1.26 (m, 4H), 1.16(s, 6H). MS (ES⁺, m/z): (M+H)⁺: 450.6.

99.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-99)

Synthesis ofN′-(5-bromopyridin-2(1H)-ylidene)-3-(trifluoromethoxy)benzohydrazide

To mixture of hydrazinepyridine (1.4 grams, 7.45 mmol) in ACN (15 mL)was added acid chloride (1.672 grams, 7.45 mmol), and the mixture wasstirred at r.t. overnight. After stirring overnight, filtration got thepure white solid and dried for the next step directly (2.60 grams, 6.91mmol, 93% yield).

Synthesis of6-bromo-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridine

To a solution ofN′-(5-bromopyridin-2(1H)-ylidene)-3-(trifluoromethoxy)benzohydrazide(1.0 gram, 2.66 mmol) AcOH 10 mL and 0.5 mL 4M HCl in dioxane were addedand the mixture was heated to 180° C. for 1 h. TLC indicated thereaction processed almost completely. After removed the solvent, theresidue was dissolved in DCM and washed with saturated NaHCO₃. Driedwith Na₂SO₄ and concentrated for chromatography purification (ethylacetate/hexane, 0-40%). White solid (0.40 grams, 1.117 mmol, 42% yield)was obtained as pure product.

¹H-NMR (CD₃OD/400 MHz): δ 8.67 (m, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.83 (s,1H), 7.79 (m, 1H), 7.77 (m, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.56 (d, J=9.6Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 358.3.

A solution of triazolopyridine (0.15 g, 0.419 mmol) and4-methoxycyclohexanaime (0.5 mL) in toluene 0.5 mL was added cesiumcarbonate (0.273 g, 0.838 mmol), rac-BINAP (78 mg, 0.127 mmol) andPd(OAc)₂ (19 mg, 0.084 mmol) and the mixture was heated at 100° C. for16 h under microwave irradiation. The resulting dark brown solution wascooled down and was concentrated under reduced pressure. The solid wasfurther purified by using combiflash chromatography (12 g column),eluent: 0-10% methanol/DCM and obtained product4-(Methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(40 mg, 0.098 mmol, 24% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.85 (d, J=8.0 Hz, 1H), 7.78 (s, 1H), 7.74 (t,J=8.4 Hz, 1H), 7.57 (d, J=10.0 Hz, 1H), 7.50 (d, J=9.2 Hz, 1H), 7.35 (s,1H), 7.16 (d, J=10.0 Hz, 1H), 3.24 (m, 1H), 3.12 (m, 1H), 2.10 (m, 4H),1.31 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 407.5.

100.1-Methyl-4-((3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexanol(EX. 8-100)

A solution of triazolopyridine (0.20 g, 0.558 mmol) and4-amino-1-methylcyclohexanol (0.216 g, 1.675 mmol) in dioxane 0.5 mL wasadded cesium carbonate (0.546 g, 1.675 mmol), rac-BINAP (104 mg, 0.168mmol) and Pd(OAc)₂ (25 mg, 0.112 mmol) and the mixture was heated at100° C. for 16 h under microwave irradiation. The resulting dark brownsolution was cooled down and was concentrated under reduced pressure.The solid was further purified by using combiflash chromatography (12 gcolumn), eluent: 0-10% methanol/DCM and obtained product1-Methyl-4-((3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)cyclohexanol(23 mg, 0.057 mmol, 10% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.85 (d, J=8.0 Hz, 1H), 7.76 (m, 2H), 7.74 (d,J=9.6 Hz, 1H), 7.50 (d, J=9.2 Hz, 1H), 7.35 (s, 1H), 7.21 (d, J=10.0 Hz,1H), 3.23 (m, 1H), 1.98 (m, 2H), 1.70 (m, 2H), 1.49 (m, 4H), 1.24 (s,6H). MS (ES⁺, m/z): (M+H)⁺: 407.5.

101.4-((3-(3-(Trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-101)

A solution of triazolopyridine (0.10 g, 0.279 mmol) and4-amino-cyclohexylpropanol (0.220 g, 1.396 mmol) in dioxane 0.5 mL wasadded cesium carbonate (0.182 g, 0.558 mmol), rac-BINAP (52 mg, 0.084mmol) and Pd(OAc)₂ (13 mg, 0.056 mmol) and the mixture was heated at100° C. for 16 h under microwave irradiation. The resulting dark brownsolution was cooled down and was concentrated under reduced pressure.The solid was further purified by using preparative HPLC and obtainedproduct4-((3-(3-(Trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)cyclohexyl)propan-2-ol.

¹H-NMR (CD₃OD/400 MHz): δ 7.83 (m, 1H), 7.72 (m, 2H), 7.70 (d, J=8.4 Hz,1H), 7.48 (m, 1H), 7.32 (s, 1H), 7.19 (d, J=8.6 Hz, 1H), 3.26 (m, 1H),2.25 (m, 2H), 1.93 (m, 2H), 1.29 (m, 4H), 1.17 (s, 6H). MS (ES⁺, m/z):(M+H)⁺: 435.6.

102.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-102)

A solution of triazolopyridine (0.075 g, 0.209 mmol) andtetrahydro-2H-pyran-4-amine (0.5 mL) in toluene 0.5 mL was added cesiumcarbonate (0.136 g, 0.419 mmol), rac-BINAP (36 mg, 0.063 mmol) andPd(OAc)₂ (9.4 mg, 0.042 mmol) and the mixture was heated at 100° C. for16 h under microwave irradiation. The resulting dark brown solution wascooled down and was concentrated under reduced pressure. The solid wasfurther purified by using combiflash chromatography (12 g column),eluent: 0-10% methanol/DCM and obtained productN-(tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(30 mg, 0.079 mmol, 38% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.84 (d, J=8.0 Hz, 1H), 7.77 (s, 1H), 7.73 (t,J=8.0 Hz, 1H), 7.59 (d, J=10.0 Hz, 1H), 7.50 (m, 1H), 7.40 (s, 1H), 7.18(d, J=9.6 Hz, 1H), 3.94 (m, 2H), 3.47 (m, 2H), 3.38 (m, 1H), 2.00 (m,2H), 1.52 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 379.5.

103.4-(((3-(3-(Trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-103)

A solution of triazolopyridine (0.20 g, 0.558 mmol) and4-(aminomethyl)tetrahydro-2H-thiopyran-1,1-dioxane (0.220 g, 1.396 mmol)in dioxane 0.5 mL was added cesium carbonate (0.546 g, 1.765 mmol),rac-BINAP (104 mg, 0.168 mmol) and Pd(OAc)₂ (25 mg, 0.112 mmol) and themixture was heated at 100° C. for 16 h under microwave irradiation. Theresulting dark brown solution was cooled down and was concentrated underreduced pressure. The solid was further purified by using preparativeHPLC and obtained product4-(((3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide.

¹H-NMR (CD₃OD/400 MHz): δ 7.92 (d, J=8.0 Hz, 1H), 7.85 (s, 1H), 7.79 (m,2H), 7.59 (m, 2H), 7.49 (s, 1H), 3.09 (m, 6H), 2.20 (m, 2H), 1.95 (m,1H), 1.83 (m, 2H). MS (ES⁺, m/z): (M+H)⁺: 441.4.

104.4-((3-(3-(Trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexanol(EX. 8-104)

To a solution of4-(methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(0.030 gram, 0.074 mmol) in DCM 1.0 mL was added TMSI 0.2 mL. Themixture was stirred at rt. for overnight. TLC indicated pretty cleanreaction. After removal the solvent completely, then the residue wasquenched with NaHCO₃ saturated aqueous solution. Chromatography onsilica gel got pure product (0.020 gram, 0.051 mmol, 69% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.95 (d, J=8.0 Hz, 1H), 7.87 (m, 3H), 7.73 (d,J=9.2 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.50 (s, 1H), 3.60 (m, 1H), 3.21(m, 1H), 2.10 (m, 2H), 1.98 (m, 2H), 1.37 (m, 4H). MS (ES⁺, m/z):(M+H)⁺: 393.5.

105.4-(cis-Bromocyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-105)

To a solution of4-(trans-methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(0.020 gram, 0.049 mmol) in DCM 1.0 mL was added TMSBr 0.25 mL withice-water cooling. The mixture was stirred at rt. for overnight. TLCindicated pretty clean reaction. After removal the solvent completely,then the residue was quenched with NaHCO₃ saturated aqueous solution.Chromatography on silica gel got pure product (0.009 gram, 0.020 mmol,40% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.84 (d, J=8.0 Hz, 1H), 7.77 (s, 1H), 7.73 (t,J=8.0 Hz, 1H), 7.58 (d, J=10.0 Hz, 1H), 7.50 (m, 1H), 7.36 (s, 1H), 7.21(d, J=10.0 Hz, 1H), 4.55 (m, 1H), 3.22 (m, 1H), 2.12 (m, 2H), 1.99 (m,2H), 1.86 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 455.4.

106.4-(trans-Methoxycyclohexyl)-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-106)

Synthesis of 2-hydrazinyl-5-iodopyridine

The solution of 2-chloro-5-iodopyridine (2.0 g, 20.9 mmol), anhydroushydrazine (3.3 mL, 105 mmol) in pyridine (40 mL) was heated at refluxovernight. After cooling to room temperature, the reaction mixture wasconcentrated under reduced pressure. To the residue was added 1 N sodiumhydroxide solution and it was extracted with EtOAc. The organic layerwas separated, dried (sodium sulfate) and concentrated under reducedpressure. To the residue was added hexanes, and the precipitate wascollected and dried in vacuo to afford the title compound (1.35 g, 69%yield) as off-white crystals. MS (ES⁺, m/z): 236.2 (M⁺+1).

Synthesis of5-iodo-2-(2-(3-(trifluoromethyl)benzylidene)hydrazinyl)pyridine

To a suspension of 5-iodo-2-hydrazinopyidine (1.32 g, 5.62 mmol) in EtOH(90 mL) was added 3-(trifluoromethyl)-benzaldehyde (0.98 g, 5.62 mmol).The mixture was heated at reflux for 5 h under argon. After cooling toRT, the resulting precipitate was collected by filtration and washedwith EtOH. The crude product was recrystallized from DCM and hexane toafford the title compound (1.80 g, 82% yield) as a white solid. MS (ES⁺,m/z): 392.3 (M⁺+1).

Synthesis of6-iodo-3-(3-(trifluoromethyl)phenyl)[1,2,4]triazolo[4,3-a]pyridine

To a solution of5-iodo-2-(2-(3-(trifluoromethyl)benzylidene)hydrazinyl)pyridine (1.60 g,4.09 mmol) in DCM (10 mL)/EtOH (1 mL), was added PhI(OAc)₂ (1.845 g,5.73 mmol). The mixture was stirred at RT for 5 h, then the solvent wasremoved in vacuo. The residue was dissolved in DCM and purified on SiO₂column chromatography with 80% 15% MeOH/EA in hexane to afford the titlecompound (1.21 g, 76% yield) as an off-white solid. MS (ES⁺, m/z): 390.3(M⁺+1).

Synthesis ofN-(trans-4-methoxycyclohexyl)-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine

To a suspension of6-iodo-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridine(0.078 g, 0.2 mmol) and trans-4-methoxycyclohexanamine (0.078 g, 0.6mmol) in toluene (0.5 mL) was added CuI (0.023 g, 0.12 mmol),ethane-1,2-diol (0.025 g, 0.4 mmol) and K₃PO₄ (0.085 g, 0.4 mmol). Itwas flushed with argon and heated at 90° C. overnight. The mixture wasabsorbed on SiO₂ and the major peak was washed out with 60%-100% 20%MeOH/DCM/0.2% NH₃ in hexane. Then the crude product was combined andpurified again on C18 column chromatography using 20-40% ACN in water togive the title compounds.

¹H NMR (400 MHz, CD₃COCD₃/CDCl₃): 8.00 (m, 2H), 7.66 (m, 3H), 7.38 (m,1H), 6.96 (m, 1H), 4.58 (br, 1H), 3.22 (s, 3H), 3.08 (m, 1H), 3.07 (m,1H), 2.00 (m, 2H), 1.18 (m, 4H).

¹⁹F NMR (376 MHz, CD₃COCD₃/CDCl₃): −57.9. MS (ES+, m/z): 391.5 (M⁺+1).

107.4-((3-(3-(Trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexanol(EX. 8-107)

¹H NMR (400 MHz, CD₃COCD₃): 8.23 (m, 2H), 7.87 (m, 2H), 7.59 (m, 1H),7.58 (m, 1H), 7.12 (dd, J=10.0, 2.0 Hz, 1H), 5.09 (d, J=7.6 Hz, 1H),3.57 (m, 1H), 3.30 (m, 1H), 2.16 (m, 4H), 1.30 (m, 4H). ¹⁹F NMR (376MHz, CD₃COCD₃): −63.4. MS (ES⁺, m/z): 377.5 (M⁺+1).

108.1-Methyl-4-((3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexanol(EX. 8-108)

To a suspension of6-bromo-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridine(0.190 g, 0.56 mmol) and trans-4-amino-1-methylcyclohexanol (0.072 g,0.56 mmol) in dioxane (1.5 mL) was added rac-Binap (0.104 g, 0.17 mmol),Pd(OAc)₂ (0.025 g, 0.11 mmol) and Cs₂CO₃ (0.362 g, 0.111 mmol). It wasflushed with argon then heated at 100° C. overnight. It was absorbed onSiO₂ and purified on column chromatography on SiO₂ with 40-100% 15%MeOH/EA in hexane. This product was further purified on C18 columnchromatography using 20-40% ACN in water.

¹H NMR (400 MHz, CD₃COCD₃): 8.23 (m, 2H), 7.88 (m, 2H), 7.94 (t, J=10.0Hz, 1H), 7.57 (s, 1H), 7.18 (d, J=10.0 Hz, 1H), 3.39 (m, 1H), 2.07 (m,2H), 1.70 (m, 2H), 1.55 (m, 4H), 1.21 (s, 3H). MS (ES+, m/z): 391.5(M⁺+1).

109.4-((3-(3-(Trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)-trans-cyclohexyl)propan-2-ol(EX. 8-109)

1H NMR (400 MHz, CD₃COCD₃): 8.28 (m, 2H), 7.99 (m, 2H), 7.94 (t, J=8.0Hz, 1H), 7.73 (s, 1H), 7.53 (d, J=10.0 Hz, 1H), 3.23 (m, 1H), 2.21 (m,2H), 1.93 (m, 2H), 1.31 (m, 2H), 1.25 (m, 2H), 1.17 (s, 6H). MS (ES⁺,m/z): 391.5 (M⁺+1).

110.N-(Tetrahydro-2H-pyran-4-yl)-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-110)

To a suspension of6-iodo-3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridine(0.101 g, 0.26 mmol) and tetrahydro-2H-pyran-4-amine (0.131 g, 1.30mmol) in toluene (1 mL) was added rac-Binap (0.049 g, 0.078 mmol),Pd(OAc)₂ (0.012 g, 0.052 mmol) and Cs₂CO₃ (0.169 g, 0.52 mmol). It wasflushed with argon then heated at 110° C. overnight. It was absorbed onSiO₂ and purified on column chromatography on SiO₂ with 60% 15% MeOH/EAin hexane. This product was further purified on C18 columnchromatography using 20-40% ACN in water.

¹H NMR (400 MHz, CD₃COCD₃): 8.24 (m, 2H), 7.85 (m, 2H), 7.63 (m, 1H),7.60 (d, J=10.0 Hz, 1H), 7.15 (d, J=10.0 Hz, 1H), 5.21 (br, 1H), 3.88(m, 2H), 3.50 (m, 1H), 3.43 (m, 2H), 1.95 (m, 2H), 1.49 (m, 2H). ¹⁹F NMR(376 MHz, CD₃COCD₃): −63.4. MS (ES+, m/z): 363.5 (M⁺+1).

111.4-(((3-(3-(Trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)amino)methyl)tetrahydro-2H-thiopyran1,1-dioxide (EX. 8-111)

¹H NMR (400 MHz, CD₃COCD₃): 8.27 (m, 2H), 7.94 (m, 1H), 7.90 (m, 1H),7.84 (d, J=9.6 Hz, 1H), 7.69 (s, 1H), 7.39 (d, J=9.6 Hz, 1H), 3.18 (d,J=6.8 Hz, 2H), 3.09 (m, 2H), 3.01 (m, 2H), 2.25 (m, 2H), 2.15 (m, 1H),1.85 (m, 2H). MS (ES⁺, m/z): 425.5 (M⁺+1).

112.2-((3-(3-(trifluoromethyl)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-yl)oxy)ethanol(EX. 8-112)

¹H NMR (400 MHz, CD₃COCD₃): 8.20 (m, 2H), 8.00 (m, 1H), 7.83 (m, 2H),7.74 (m, 1H), 7.25 (m, 1H), 4.15 (m, 2H), 3.97 (m, 2H). ¹⁹F NMR (376MHz, CD₃COCD₃): −63.1. MS (ES+, m/z): 324.4 (M⁺+1).

113.4-(trans-Methoxycyclohexyl)-3-(3-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(EX. 8-113)

Synthesis of N′-(5-bromopyridin-2(1H)-ylidene)-3-chlorobenzohydrazide

To mixture of hydrazinepyridine (2.0 grams, 10.64 mmol) in ACN (15 mL)was added acid chloride (1.86 grams, 10.64 mmol), and the mixture wasstirred at r.t. overnight. After stirring overnight, filtration got thepure white solid and dried for the next step directly (3.0 grams, 9.19mmol, 86% yield). MS (ES⁺, m/z): (M+H)⁺: 326.3.

Synthesis of 6-bromo-3-(3-chlorophenyl)-[1,2,4]triazolo[4,3-a]pyridine

To a solution ofN′-(5-bromopyridin-2(1H)-ylidene)-3-chlorobenzohydrazide (3.0 grams,9.19 mmol) AcOH 10 mL and 0.5 mL 4M HCl in dioxane were added and themixture was heated to 180° C. for 1 h. TLC indicated the reactionprocessed almost completely. After removed the solvent, the residue wasdissolved in DCM and washed with saturated NaHCO₃. Dried with Na₂SO₄ andconcentrated for chromatography purification (ethyl acetate/hexane,0-40%). White solid (0.30 grams, 0.972 mmol, 11% yield) was obtained aspure product.

¹H-NMR (CD₃OD/400 MHz): δ 8.65 (m, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.80 (s,1H), 7.77 (m, 1H), 7.75 (m, 1H), 7.58 (d, J=8.6 Hz, 1H), 7.56 (d, J=9.2Hz, 1H). MS (ES⁺, m/z): (M+H)⁺: 308.3.

A solution of triazolopyridine (0.13 g, 0.419 mmol) and4-methoxycyclohexanaime (0.5 mL) in toluene 0.5 mL was added cesiumcarbonate (0.273 g, 0.838 mmol), rac-BINAP (78 mg, 0.127 mmol) andPd(OAc)₂ (19 mg, 0.084 mmol) and the mixture was heated at 100° C. for16 h under microwave irradiation. The resulting dark brown solution wascooled down and was concentrated under reduced pressure. The solid wasfurther purified by using combiflash chromatography (12 g column),eluent: 0-10% methanol/DCM and obtained product4-(Methoxycyclohexyl)-3-(3-(trifluoromethoxy)phenyl)-[1,2,4]triazolo[4,3-a]pyridin-6-amine(50 mg, 0.140 mmol, 33% yield).

¹H-NMR (CD₃OD/400 MHz): δ 7.83 (s, 1H), 7.75 (d, J=6.8 Hz, 1H), 7.57 (m,3H), 7.35 (s, 1H), 7.14 (d, J=10.0 Hz, 1H), 3.20 (m, 1H), 3.12 (m, 1H),2.07 (m, 4H), 1.29 (m, 4H). MS (ES⁺, m/z): (M+H)⁺: 357.5.

Example 2 Pim Kinase Activity Assays 1. Pim-1 Kinase Inhibition Assay

One illustrative manner in which Pim-1 kinase activity can be determinedis by quantifying the amount of ATP remaining in solution after an invitro Pim-1 kinase reaction. The Kinase-Glo Assay Kit (Promega, Inc.,Madison, Wis.) allows this. The amount of ATP remaining in the solutionafter the kinase reaction serves as a substrate for the luciferase tocatalyze luciferin to oxyluciferin plus one photon of light. Thus, theluminescent signal read by the Luminoskan Ascent Instrument (ThermoElectron Corp., Milford, Mass.) correlates with the amount of ATPpresent after the kinase reaction and inversely correlates with theamount of kinase activity. This assay is efficient at determining theIC₅₀ values of kinase inhibitors against the Pim-1 kinase. These assaysare set up in duplicate 50 ul volumes in white, flat bottom 96 wellplates. Inhibitors are added to the solution of 1× kinase buffer, 10 uMATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, andwater in serial dilutions ranging from micromolar to nanomolarconcentrations. This solution is incubated at 30 degrees Celsius at 360rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagentis added to each well, including all positive and negative controlwells, and incubated at room temperature for 15 minutes. The plate isthen read by the Luminoskan Ascent instrument and the results displayedwith the Ascent Software version 2.6. The IC₅₀ values can then becalculated for each inhibitor tested.

Alternatively, Pim-1 kinase activity can be determined by quantifyingthe phosphorylation of a known Pim-1 substrate in another in vitroassay. The Z-Lyte Protein Kinase Assay Kit (Invitrogen, Madison Wis.)allows this, using Fluorescent Resonance Energy Transfer (FRET)procedure. Briefly, a known Pim-1 substrate (Serine-Threonine Substratefrom Invitrogen), which bears two fluorophores at opposing ends(coumarin and fluorescein) is incubated with Pim-1 enzyme and apotential inhibitor. Following this, the kinase reaction is stopped, anda development reagent is added. This reagent, a protease, will cleaveonly unphosphorylated substrate, separating the two fluorophores andreducing the amount of FRET which can occur between them. FRET can thenbe measured using a spectrophotometer, such as the Gemini EM (MolecularDevices). A reduction in FRET is indicative of an active inhibitor.

Alternatively, PIM kinase IC₅₀ and K_(i) determinations were determinedat Reaction Biology (Malvern, Pa.). For K_(i) determination, PIM-1 wereincubated with 10-dose, 3-fold serial dilutions of compound startingwith 10 μM using 5 different concentrations of ATP (25, 50, 100, 250 and500 μM ATP for PIM-1; 5, 10, 20, 50 and 100 μM ATP for PIM-2 and PIM-3),and the activity was measured at 0, 5, 10, 15, 20, 30, 45, 60, 75, 90,105 and 120 minutes. The data was analyzed in a Michaelis-Menton plot todetermine apparent K_(m) and K_(i) values using GraFit software using amixed inhibition equation for global fit.

2. Cell-Based Pim-1 Kinase Inhibitor Assays

Cell culture-based assays can be used to evaluate the ability ofcompounds of the invention to inhibit one or more cellular activities,such as cancer cell growth and/or survival. Numerous cancer cell linescan be obtained from the American Type Culture Collection (ATCC) andother sources. Briefly, cells are seeded into 96-well, tissue-culturetreated, opaque white plates (Thermo Electron, Vantaa, Finland), atbetween 5000 and 10000 cells per well, depending on the speed of cellproliferation, in 100 μl of appropriate growth medium (determined by theATCC). Cells are then exposed to the appropriate concentration of drugor an equal amount of DMSO (drug diluent) and allowed to grow in itspresence for 96 hours. Following this, 100 μl of Cell-Titer-Glo reagent(Promega, Inc., Madison, Wis.) is added to each well. Plates are thenshaken for 2 minutes at room temperature to allow for cell lysis andincubated for 10 minutes at room temperature to stabilize theluminescent signal. Similar to the Kinase-Glo assay reagent fromPromega, this reagent contains both luciferase enzyme and its substrateluciferin. Luciferase, activated by ATP in the cell lysate, catalyzesthe conversion of luciferin to oxyluciferin, a reaction which produceslight. The amount of light produced is proportionate to the amount ofATP in the cell lysate, which is itself proportional to cell number andgives an index of cellular proliferation.

In order to detect specific inhibition of Pim-1 enzyme in cell culture,a Western blot assay will also be performed. For this, cells which havebeen treated with a potential Pim-1 inhibitor are lysed with a bufferspecific for the isolation and preservation of proteins (1% NonidetP-40, 150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA, 1:500 ProteaseInhibitor Cocktail III [Calbiochem], 100 mM NaF, 100 mM SodiumOrthovanadate). The protein concentration in these lysates is thenquantified using the BCA Protein Assay Kit (Pierce). Known amounts ofprotein, e.g. 10 μg, are loaded onto 12% SDS-polyacrylamide gels and aresubjected to reducing, denaturing SDS-PAGE. Electrophoresed proteins aretransferred to a nitrocellulose membrane, which is then probed withantibodies to p-21 and phospho (Thr 145) p-21. As Threonine-145 of thep-21 protein is a substrate for Pim-1, measuring the amount ofphosphorylation at this site in treated cells should provide a means bywhich to evaluate the efficacy of our Pim-1 inhibitors.

3. Pim-1 Kinase Specific Activity Data

Using procedures essentially as described above, illustrative compoundswere tested for inhibition of Pim-1 kinase activity. IC₅₀ values weredetermined for illustrative compounds against Pim-1 kinase, using thePromega Kinase-Glo assay, the results for which are summarized in TablesIV and V below.

TABLE IV Compound IC₅₀ on Pim1. EX. IC₅₀ (nM) EX. IC₅₀ (nM) 8-1 13 8-218 8-3 30 8-4 7 8-5 36 8-6 30 8-7 10 8-8 4 8-9 13 8-10 9 8-11 8 8-12 818-13 58 8-14 1190 8-15 2830 8-16 503 8-17 173 8-18 53 8-19 13 8-20 168-21 46 8-22 41 8-23 1630 8-24 34 8-25 30 8-26 28 8-27 23 8-28 553 8-295 8-30 103 8-31 1 8-32 115 8-33 33 8-34 241 8-35 7 8-36 145 8-37 4 8-3818 8-39 48 8-40 200 8-41 226 8-42 1970 8-43 523 8-44 153 8-45 16 8-46 278-47 204 8-48 197 8-49 378 8-50 633 8-51 148 8-52 5 8-53 132 8-54 258-55 54 8-56 17 8-57 66 8-58 149 8-59 111 8-60 NA 8-61 237 8-62 378 8-6323 8-64 68 8-65 186 8-66 164 8-67 45 8-68 4 8-69 24 8-70 8 8-71 12 8-7258 8-73 17 8-75 56 8-76 687 8-77 14 8-78 206 8-79 2 8-80 52 8-82 34 8-8389 8-84 239 8-85 19 8-86 14 8-88 69 8-92 38 8-95 24 8-96 94 8-97 1688-98 470

TABLE V Compound IC₅₀ on Pim1. EX. IC₅₀ (nM) EX. IC₅₀ (nM) 8-99 83 8-1001644 8-101 488 8-102 3250 8-103 3805 8-104 684 8-105 2490 8-106 5188-107 3948 8-108 70 8-109 3870 8-110 3165 8-111 >30 μM 8-112 >30 μM8-113 722

Example 3 hERG Activity Assays

Representative compounds were tested for hERG activity using the FastPatch assay available from WuXiApptec (Shanghai China). The hERGactivity for representative compounds is provided in Table VI below.

TABLE VI hERG Activity of Representative Compounds Compound hERG IC50(μM) 8-37 >30 8-35 30 8-64 >30 8-39 >30 8-31 >30 8-30 12 8-29 108-10 >30 8-9 26 8-11 >30 8-63 >30 8-54 >30 8-56 >30 8-46 >30 8-1 16 8-1310

Example 4 Cell Based Assays

The cellular potency of a representative compound (8-31) was determinedby measuring its effect on baseline phosphorylation of BAD (BCL-2antagonist of cell death), a known substrate of PIM, on serine 112 byover-expression of PIM-1 and BAD in HEK-293 cells. Over-expression ofthe catalytically inactive mutant PIM-1 (K67M) did not increasephosphorylation of BAD compared to BAD transfection alone (data notshown), and was used as a negative control to subtract BADphosphorylation by cellular kinases other than PIM-1.

Compound 8-31 demonstrated potent PIM-1 specific cellular activity inthe PIM-1/BAD over-expression system with an average EC₅₀=67 nM (FIG.1).

Example 5 Tumor Xenografts

In an effort to demonstrate tumorigenicity of the PIM kinases and toevaluate representative compounds (e.g., EX. 8-31) in PIM-driven tumorxenografts, PIM-1 and PIM-2 over-expression cell lines were developed.The prostate cancer cell line 22RV1 engineered to over-express PIM-1 aspreviously described (Mumenthaler, et al, Mol. Cancer Ther., 2009,8(10)2882-93) was evaluated in vivo. A second model engineered toover-express PIM-2 was established using the NIH-3T3 mouse fibroblastcell line. Both cell backgrounds displayed increased expression of PIM-1or PIM-2 kinase by Western blot, and significantly enhanced the observedgrowth rate in cell culture (FIG. 2). PIM-1 overexpression in 22RV1cells significantly enhanced subcutaneous tumor growth compared to theparental cell line when grown as mouse xenografts (22RV1/PIM-1+vehiclevs. 22RV1+vehicle), and the growth was significantly reduced byadministration of compound 8-31 (22RV1/PIM-1+vehicle vs.22RV1/PIM-1+compound 8-31) (FIG. 3A). PIM-1 over-expression demonstrateda similar effect on xenograft growth in the presence or absence ofvehicle treatment (data not shown). No significant changes in bodyweight were observed in mice from any group. Similarly, PIM-2over-expression in NIH-3T3 cells significantly induced subcutaneoustumor growth compared to the parental cell line when grown as mousexenografts (NIH-3T3/PIM-2+vehicle vs. NIH-3T3+vehicle), and the growthwas significantly inhibited by compound 8-31 (NIH-3T3/PIM-2+vehicle vs.NIH-3T3/PIM-2+compound 8-31) (FIG. 3B). Likewise, PIM-2 over-expressiondemonstrated a similar effect on xenograft growth with or withoutvehicle administration (data not shown). No significant changes in bodyweight were observed in any group.

Example 6 Translational Models

Translational models were also explored for PIM dependency using shRNAknockdown. The UM-UC-3 urinary epithelial bladder carcinoma cell linewas used to verify and validate dependency on PIM-1 for growth. PIM-1mRNA was significantly reduced using two independent shRNAs targetingPIM-1 compared to the non-target shRNA control (FIG. 4A). Further, PIM-1protein was reduced using PIM-1 shRNA compared to non-target shRNA, and2D-colony growth was markedly reduced with PIM-1 knockdown (FIG. 4A),comparable to a previous report in the literature (Guo, S (2010) J ExpClin Cancer Res 29:161). Reduction of PIM-1 mRNA and growth also wereobserved using both PIM-1 and PIM-2 shRNA in the androgen-independentprostate carcinoma cell line PC-3 (data not shown). Finally, compound8-31 reduced colony growth of UM-UC-3 and PC-3 cells (FIGS. 4B and 4C),confirming the PIM-1 dependent growth for both cell lines.

Example 7 Growth Inhibition of Mouse Xenograft Tumors

Compound 8-31 was tested for inhibiting the growth of established mousexenograft tumors using the UM-UC-3 and PC-3 solid tumor cell lines thatwere tested in vitro. Oral dosing of 200 mg/kg of compound 8-31significantly reduced both UM-UC-3 and PC-3 tumor growth measured byvolume (caliper) and by final tumor weight, with no significant changesin body weight or gross adverse toxicity (FIG. 5). In addition, dosingof compound 8-31 at 125 mg/kg in each model led to significant tumorgrowth inhibition, albeit less than that observed with 200 mg/kg,indicating a dose-response effect and a therapeutic window (data notshown). Pharmacokinetic studies of the PC-3 tumor-burdened mice revealedthat 200 mg/kg compound 8-31 dosing led to plasma and tumor tissuelevels above the in vitro proliferation EC₅₀ (data not shown).

Example 8 Hematological Cancer Cell Screening

PIM kinases are implicated in the survival of hematological cancers,therefore compound 8-31 was screened against a panel of hematologicalcell lines using an in vitro viability assay. Over 35 cell lines weretested, and the most sensitive cell lines from various indications wereselected and verified by multiple EC₅₀ determinations (FIG. 6). Compound8-31 displayed nearly equivalent EC₅₀ values for the most sensitivehematological cell lines compared to the bladder line, UM-UC-3, in therange of 1-2 μM. Anti-proliferative activity using PIM-2 shRNA againstFLT3-ITD driven leukemia lines has been observed (Adam (2006) Cancer Res66:3828-35 data not shown). Notably, MV-4-11 and MOLM-13 wereparticularly sensitive to SGI-9481, while not wishing to be bound bytheory, it is believed that this is potentially due to the combinationof PIM kinase and FLT3 inhibition, although the activity could bepossibly attributed to the FLT3 activity of the compound alone. Whilenot wishing to be bound by theory, the inhibitory activity of compound8-31 (and other compounds of the invention) against multiple members ofthe phosphatidyl inositol-3-kinase (PI3K) family observed in theselectivity study may impart additional advantage when targetingdifferent tumor types

Example 9 Mouse Xenograft Studies

Compound 8-31 demonstrated significant tumor growth inhibition in mousexenograft studies with ML-2 cells (wild-type FLT3) (FIG. 7A), similar tothe level of reduction observed in the solid tumor models. Treatment ofestablished xenografts of MV-4-11 (FLT3-ITD) with compound 8-31 alsoinduced regressions (FIG. 7B). While not wishing to be bound by theory,these data indicate that AML models are sensitive to compound 8-31,where FLT3-ITD status enhanced the antitumor effects observed.

1. A compound having one of the following structures (I), (II) or (III):

or a stereoisomer, prodrug, tautomer or pharmaceutically acceptable saltthereof, wherein: X is a direct bond, NH, N(alkyl), S, O, SO or SO₂; Ris H, —OH, halo, alkyl, haloalkyl, alkoxy, haloalkoxy, —NH₂, —NH(alkyl),—N(alkyl)₂, or —CN; R₁ is optionally substituted carbocycle, optionallysubstituted heterocycle or R₁ has the following structure:

where R₁′ is at, each occurrence, independently selected from hydrogencyano, alkyl, alkoxy, halo, haloalkyl, haloalkoxy, —OCF₃, —OCHF₂, —CF₃,—OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ and —N(CH₃)₂ and p is 1, 2 or3 R₂ is

 —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclohexyl, —SO₂—CH₃,—(CH₂)_(n)-piperonyl, —(CH₂)_(n)-piperidin-2-onyl,—(CH₂)_(n)-piperazinyl, —(CH₂)_(n)-thiophenyl, —(CH₂)_(n)-pyridyl,—(CH₂)_(n)-pyrimidyl, —(CH₂)_(n)-thiomorpholinylsulfone,—(CH₂)_(n)-phenyl, (e.g., unsubstituted phenyl)—(CH₂)_(n)-tetrahydropyranyl, —(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)_(n)-morpholinyl,—(CH₂)—OCH₃, —(CH₂)—OH, —(CH₂)—C(CH₃)₂OH or —(CH₂)_(n)N(CH₃)₂, where Wis —O—, —S(O)_(z)— or >C(R⁹)[(CR¹⁰R¹¹)_(y)R¹²]; R³, R⁴, R⁷, R⁹, R¹⁰ andR¹¹ are, at each occurrence, independently H or alkyl; R¹² is —OH, —CNor alkoxy; m is 1, 2, 3, 4, 5 or 6; n is 0, 1, 2, 3 or 4; y and z areeach independently 0, 1 or 2 and each of the above moieties areoptionally substituted with one or more substituents; or R₂ has one ofthe following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionallysubstituted with one or more substituents, and wherein when R₂ is—(CH₂)_(n)-tetrahydropyranyl, then R₁ is not phenyl substituted withcarboxy.
 2. The compound of claim 1, wherein the compound has structure(I).
 3. The compound of claim 1, wherein the compound has structure(II).
 4. The compound of claim 1, wherein the compound has one of thefollowing structures (I-D) or (II-D):

wherein: X is —N(R⁸)— or —O—; W is —O—, —S(O)_(z)— or

R is H, —OH, halo, alkyl, haloalkyl, alkoxy, haloalkoxy, —N(R⁸)₂, or—CN; R³, R⁴, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are, at each occurrence,independently H or alkyl; R⁵ is halo, haloalkyl or haloalkoxy; R⁶ is H,—OH, alkyl or alkoxy; R¹² is —OH, —CN or alkoxy; m is 1, 2, 3, 4, 5 or6; n is 0, 1, 2, 3 or 4; and y and z are each independently 0, 1 or 2.5. The compound of claim 1, wherein R⁶ is H.
 6. The compound of claim 1,wherein R is H.
 7. The compound of claim 1, wherein R⁵ is at the metaposition and the compound has one of the following structures (I-Da) or(II-Da):


8. The compound of claim 1, wherein m is 3 or
 4. 9. The compound ofclaim 1, wherein m is
 4. 10. The compound of claim 1, wherein thecompound has one of the following structures (IDb) or (IIDb):


11. The compound of claim 1, wherein R⁷ is H.
 12. The compound of claim1, wherein n is 0 or
 1. 13. The compound of claim 1, wherein n is
 0. 14.The compound of claim 1, wherein at least one of R³ or R⁴ is H.
 15. Thecompound of claim 1, wherein each of R³ and R⁴ is H.
 16. The compound ofclaim 1, wherein R⁵ is —OCF₃, —CF₃, Cl or F.
 17. The compound of claim1, wherein R¹² is —OH, —CN or —OCH₃.
 18. The compound of claim 1,wherein R⁹ is H or methyl.
 19. The compound of claim 1, wherein at leastone of R¹⁰ or R¹¹ is methyl.
 20. The compound of claim 1, wherein eachof R¹⁰ and R¹¹ is methyl.
 21. The compound of claim 1, wherein y is 0or
 1. 22. The compound of claim 1, wherein W is —O—, —S(O)₂—, —CH(OH)—,—CH(CN)—, —C(CH₃)(OH)—, —CH(OCH₃)— or —CH[C(CH₃)₂OH]—.
 23. The compoundof claim 1, wherein X is —NH—.
 24. The compound of claim 1, wherein X is—O—.
 25. The compound of claim 1, wherein the compound has a hERG IC₅₀activity of 10 μM or more.
 26. The compound of claim 1, wherein thecompound has a hERG IC₅₀ activity of 30 μM or more.
 27. The compound ofclaim 1, wherein the compound has one of the following structures:


28. The compound of claim 1, wherein the compound has a structureselected from any one of the compounds in Table I.
 29. The compound ofclaim 1, wherein the compound has a structure selected from any one ofthe compounds in Table II.
 30. The compound of claim 1, wherein thecompound has structure (III).
 31. The compound of claim 30, wherein: Xis a direct bond, NH, N(alkyl), S, O, SO or SO₂; R is H, —OH, —CN, halo,alkyl, haloalkyl, alkoxy or haloalkoxy; R₁ is carbocycle, substitutedcarbocycle, heterocycle, or substituted heterocycle; or a structureselected from:

where R₁′ is a p, o or m substitution with one or more occurrences ofhalo, —OCF₃, —OCHF₂, —CF₃, —CN, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃,—NHSO₂CH₃ or —N(CH₃)₂, —CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂; R₂ is—(CH₂)_(n)-cyclopropyl, —(CH₂)_(n)-cyclobutyl, —(CH₂)_(n)-cyclopentyl,—(CH₂)_(n)-cyclohexyl, —SO₂—CH₃, —SO₂—(CH₂)—CH₃, —(CH₂)_(n)-piperonyl,—(CH₂)_(n)-piperidyl, —(CH₂)-piperidin-2-only, —(CH₂)_(n)-piperazinyl,—(CH₂)_(n)-furyl, —(CH₂)_(n)-thiophene, —(CH₂)_(n)-pyridyl,—(CH₂)_(n)-pyrimidyl, —(CH₂)_(n)-thiomorpholinylsulfone,—(CH₂)_(n)-phenyl, —(CH₂)_(n)-tetrahydropyranyl,—(CH₂)_(n)-tetrahydrothiopyranyl,—(CH₂)_(n)-tetrahydrothiopyranylsulfone, —(CH₂)—OCH₃, —(CH₂)—OH,—(CH₂)—N(CH₃)₂, —(CH₂)—CH(CH₃)₂OH or —(CH₂)_(n)N(CH₃)₂, where n is 0, 1,2, 3 or 4 and each of the above moieties are optionally substituted withone or more substituents; or R₂ is a structure selected from:

where L₁ is optional and, if present, NH, S, O, SO or SO₂, N-alkyl, C═O,C═O(NH); R₃ is one or more optional substituents; and Cycl₁ is acarbocycle, substituted carbocycle, heterocycle or substitutedheterocycle.
 32. The compound of claim 30, wherein R is hydrogen. 33.The compound of claim 30, wherein X is NH.
 34. The compound of claim 30,wherein R₁ is substituted phenyl having at least one p, o or msubstituent selected from halo, —OCF₃, —OCHF₂, —CF₃, —CN, —OCH₃, and—OH.
 35. The compound of claim 30, wherein R₁ is selected from:

where R₁′ is a p, o or m substitution with one or more occurrences ofhalo, —OCF₃, —OCHF₂, —CF₃, —OCH₃, —NH₂, —NO₂, —OH, —COCH₃, —NHSO₂CH₃ or—N(CH₃)₂, —CONH₂, —CO—NH-alkyl, —CO—N-alkyl₂.
 36. The compound of claim30, wherein R₁ is selected from:


37. The compound of claim 30, wherein R₂ is a structure selected from onof the following structures:

where n is 0, 1, 2, 3 or 4 and each of the above moieties are optionalsubstituted with one or more substituents.
 38. The compound of claim 30,wherein R₂ is selected from:


39. The compound of claim 30, wherein X is NH, R is H, R₁ is substitutedphenyl, and R₂ is selected from:


40. The compound of claim 30, wherein the compound has one of thefollowing structures:


41. A pharmaceutical composition comprising the compound of claim 1 anda pharmaceutically acceptable carrier.
 42. A method for treating a Pimkinase-mediated disease, the method comprising administering to asubject in need thereof a therapeutically effective amount of thecomposition of claim
 41. 43. The method of claim 42, wherein the Pimkinase mediated disease is a Pim kinase-expressing cancer.
 44. Themethod of claim 43, wherein the cancer is bladder cancer.
 45. The methodof claim 43, wherein the cancer is prostate cancer.
 46. The method ofclaim 43, wherein the cancer is a hematological malignancy.
 47. Themethod of claim 46, wherein the hematological malignancy is acutemyeloid leukemia.
 48. The method of claim 42, wherein the Pimkinase-mediated disease is an autoimmune or inflammatory disease.